Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ß3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ß3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ß3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

A highly dynamic F-actin network regulates transport and recycling of micronemes in Toxoplasma gondii vacuoles.

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.

Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells.

The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGß1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.