Septins are a large, evolutionarily conserved family of GTPases that form hetero-oligomers and interact with the actin-based cytoskeleton and microtubules. They are involved in scaffolding functions, and form diffusion barriers in budding yeast, the sperm flagellum and the base of primary cilia of kidney epithelial cells. We investigated the role of septins in the primary cilium of retinal pigmented epithelial (RPE) cells, and found that SEPT2 forms a 1∶1∶1 complex with SEPT7 and SEPT9 and that the three members of this complex co-localize along the length of the axoneme. Similar to observations in kidney epithelial cells, depletion of cilium-localized septins by siRNA-based approaches inhibited ciliogenesis. MAP4, which is a binding partner of SEPT2 and controls the accessibility of septins to microtubules, was also localized to the axoneme where it appeared to negatively regulate ciliary length. Taken together, our data provide new insights into the functions and regulation of septins and MAP4 in the organization of the primary cilium and microtubule-based activities in cells.
αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion.
While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E‐cadherin‐mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV‐G preferentially target to sites of adhesion to E‐cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E‐cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.
Cell−cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm2/s and mobile fraction of 30−60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.
It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.
Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by epsilon-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.
As part of the E-cadherin–β-catenin–αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell–cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin–dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell–cell adhesion.
