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.
Voltage‐gated sodium channels comprise an ion‐selective α‐subunit and one or more associated β‐subunits. The β3‐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 β3‐subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3‐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 β3‐subunits. The oligomeric status of the Nav1.5 α‐subunit in vivo, with or without the β3‐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 β3‐subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest‐neighbor distances between Nav1.5 α‐subunits. However, when assayed by PLA, the β3‐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 β3‐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 β3‐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 β3‐subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.
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