Relationships between EEA1 binding partners and their role in endosome fusion

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips

AIMS: To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A).

METHODS: Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility.

RESULTS: Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips.

CONCLUSIONS: The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes.