Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with ß subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.

Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast cancer.

Phosphoinositide specificity of and mechanism of lipid domain formation by annexin A2-p11 heterotetramer.

Annexin A2 is a phospholipid-binding protein that forms a heterotetramer (annexin II-p11 heterotetramer; A2t) with p11 (S100A10). It has been reported that annexin A2 is involved in binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and in inducing membrane microdomain formation. To understand the mechanisms underlying these findings, we determined the membrane binding properties of annexin A2 wild type and mutants both as monomer and as A2t. Our results from surface plasmon resonance analysis showed that A2t and annexin A2 has modest selectivity for PtdIns(4,5)P2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys279 and Lys281, on the convex surface of annexin A2. Fluorescence microscopy measurements using giant unilamellar vesicles showed that A2t of wild type, but not (K279A)2-(p11)2 or (K281A)2-(p11)2, specifically induced the formation of 1-microm-sized PtdIns(4,5)P2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P2 selectivity of A2t and suggest that A2t may be involved in the regulation of PtdIns(4,5)P2 clustering in the cell.