CDC42EP5/BORG3 modulates SEPT9 to promote actomyosin function, migration, and invasion.

Fast amoeboid migration is critical for developmental processes and can be hijacked by cancer cells to enhance metastatic dissemination. This migratory behavior is tightly controlled by high levels of actomyosin contractility, but how it is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to migration are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for amoeboid melanoma cells to invade and migrate into collagen-rich matrices and locally invade and disseminate in vivo. Cdc42EP5 associates with actin structures, leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 affects these functions through SEPT9-dependent F-actin cross-linking, which enables the generation of F-actin bundles required for the sustained stabilization of highly contractile actomyosin structures. This study provides evidence that Cdc42EP5 is a regulator of cancer cell motility that coordinates actin and septin networks and describes a unique role for SEPT9 in melanoma invasion and metastasis

Septin Function in T Lymphocyte Crawling and Structural Integrity

T lymphocytes are components of the adaptive immune response that circulate throughout the body in search of antigen presenting cells bearing peptide-MHC complex molecules. T cells are inherently polarized with an anterior leading edge and a posterior protrusion called the uropod. While several details of T cell polarity have been established, the source and significance of this morphology is still not completely understood. Septins are components of the cytoskeleton important for structural integrity and cell cycle control. They form heteromeric complexes that interact with both actin filaments and microtubules and have diverse biological functions. Therefore, we decided to investigate the role of septins in T cell morphology.Septin complexes are found at the cell cortex and are enriched at the T cell mid-body. In order to assess the functional requirements of septins in T cells, we designed short hairpin RNAs (shRNAs) targeting Septins 1, 6, 7 and 9. Targeting of Sept7 resulted in the reduction of the entire septin complex, a result that was only observed when Sept7 was targeted. T cells lacking septin complexes have a pronounced increase in uropod length. To determine a potential mechanism for this result, we examined the activation state of myosin II in septin deficient T cells. In these cells, we did not observe any differences in the phosphorylation of either the myosin light chain or heavy chain, although myosin II activity was still required for uropod formation. Although septin deficient T cells crawled at normal velocities, they exhibited structural instabilities including membrane blebbing and the presence of excess protrusions. Septin deficient T cells also had a slight reduction in actin filaments. Therefore, it is not clear if the membrane defects we observed during crawling are from the reduction in actin filaments, reduction in the septin cytoskeleton or a combination of both. Finally, loss of septins did not affect functions that require cell polarization; however, septin deficient T cells rapidly permeated extremely small pores, consistent with the loss of a structural scaffold. Thus, septins play a critical role in T cell morphology and structural integrity and may be important for transendothelial migration

Septin Function in T Lymphocyte Crawling and Structural Integrity

T lymphocytes are components of the adaptive immune response that circulate throughout the body in search of antigen presenting cells bearing peptide-MHC complex molecules. T cells are inherently polarized with an anterior leading edge and a posterior protrusion called the uropod. While several details of T cell polarity have been established, the source and significance of this morphology is still not completely understood. Septins are components of the cytoskeleton important for structural integrity and cell cycle control. They form heteromeric complexes that interact with both actin filaments and microtubules and have diverse biological functions. Therefore, we decided to investigate the role of septins in T cell morphology.Septin complexes are found at the cell cortex and are enriched at the T cell mid-body. In order to assess the functional requirements of septins in T cells, we designed short hairpin RNAs (shRNAs) targeting Septins 1, 6, 7 and 9. Targeting of Sept7 resulted in the reduction of the entire septin complex, a result that was only observed when Sept7 was targeted. T cells lacking septin complexes have a pronounced increase in uropod length. To determine a potential mechanism for this result, we examined the activation state of myosin II in septin deficient T cells. In these cells, we did not observe any differences in the phosphorylation of either the myosin light chain or heavy chain, although myosin II activity was still required for uropod formation. Although septin deficient T cells crawled at normal velocities, they exhibited structural instabilities including membrane blebbing and the presence of excess protrusions. Septin deficient T cells also had a slight reduction in actin filaments. Therefore, it is not clear if the membrane defects we observed during crawling are from the reduction in actin filaments, reduction in the septin cytoskeleton or a combination of both. Finally, loss of septins did not affect functions that require cell polarization; however, septin deficient T cells rapidly permeated extremely small pores, consistent with the loss of a structural scaffold. Thus, septins play a critical role in T cell morphology and structural integrity and may be important for transendothelial migration

Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry

The making of a maritime explorer: the early career of Matthew Flinders

This is an Accepted Manuscript of an article published by Taylor & Francis in Journal for Maritime Research on 09/08/2016, available online: https://www.tandfonline.com/doi/full/10.1080/21533369.2016.1172839This article examines the early career of Matthew Flinders (1774–1814) to determine the ways in which his training as a navigator, hydrographer and cartographer prepared him for undertaking the first circumnavigation of Australia in the Investigator between 1801 and 1803. Two main questions are answered in relation to this theme. First, why was such a young naval officer as Flinders (he was 26) selected to lead such an important voyage of discovery? And, second, in what ways did the choice of Flinders as the commander of the Investigator reflect the training he had received? The article argues that the making of Flinders as a maritime explorer was forged through his successive nautical achievements in the 1790s, partly in relation to William Bligh’s second breadfruit voyage to the Pacific in 1791–1792 and partly in relation to voyages along the coasts of New South Wales and Van Diemen’s Land that Flinders undertook between 1795 and 1800. During that period, Flinders jointly discovered Bass Strait and circumnavigated Van Diemen’s Land: these were the two most significant geographical discoveries in Australia since the era of Captain Cook. The article shows that when he returned from Port Jackson to London in 1800, Flinders was the most experienced candidate with knowledge of Australian waters who was available to lead the Investigator expedition. It also argues that Flinders placed himself in a highly favourable position to be selected as the leader of the circumnavigation through demonstrating to Sir Joseph Banks, his patron, that he had a thorough, up-to-date knowledge of what needed to be discovered about Australia through maritime exploration. Throughout, there will be an emphasis on the nautical skills Flinders acquired rapidly as a young midshipman and lieutenant and on his determination and initiative to devote his career to Australian discovery.The research was supported by an Australian Bicentennial Fellowship, Menzies Centre for Australian Studies, King’s College, London