Robert D. Burgoyne

The Rab5 effector EEA1 is a core component of endosome docking

Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5 (refs 10,11,12,13,14,15,16,17,18,19,20). Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling

In neurons, intracellular calcium signals have crucial roles in activating neurotransmitter release and in triggering alterations in neuronal function. Calmodulin has been widely studied as a Ca2+ sensor that has several defined roles in neuronal Ca2+ signalling, but members of the neuronal calcium sensor protein family have also begun to emerge as key components in a number of regulatory pathways and have increased the diversity of neuronal Ca2+ signalling pathways. The differing properties of these proteins allow them to have discrete, non-redundant functions.

Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells

The effect of endogenous glutamate on neurite outgrowth from cerebellar granule cells in culture was examined. Neurite outgrowth was inhibited by enzymatic removal of endogenous glutamate from the culture medium. The broad‐spectrum glutamate receptor antagonist kynurenate also inhibited neurite outgrowth from granule cells in serum‐containing and serum‐free cultures; the inhibition by kynurenate was reversed by exogenous glutamate. Neurite outgrowth was inhibited to the same extent by the NMDA receptor antagonist APV. These results indicate that endogenous glutamate, possibly released by granule cells themselves, stimulated neurite outgrowth through activation of the NMDA class of glutamate receptors. Activation of NMDA receptors on developing neurons may be an important mechanism for the regulation of neuronal growth and differentiation.

The annexin family of calcium-binding proteins

The annexins are a family of calcium-binding proteins. Data from protein and cDNA sequencing have shown that at least five distinct but closely related mammalian annexins exist each of which possesses four or eight homologous internal repeats which may be calcium-and phospholipid-binding domains. The proteins are present within a wide range of tissues and cell types, with each cell type having all or a subset of the proteins. The proteins are localised on the inner surface of the plasma membrane associated with the cytoskeleton and in some cases also with intracellular structures. Some members of the family are major substrates for tyrosine and serine kinases. The precise functions of the proteins are unknown but they are likely to play important roles in cellular regulation. Previously suggested functions are inhibition of phospholipase A2, membrane-cytoskeletal linkage and control of membrane fusion events in exocytosis. It is also suggested that they may be involved in the regulation of cell surface receptors.

Protein phosphorylation and the regulation of synaptic membrane traffic.

It is well established that protein phosphorylation has an important role in synaptic plasticity. This is achieved, in part, via the presynaptic modulation of neurotransmitter release by protein kinases and protein phosphatases. In recent years, the increase in information available about proteins that are involved in synaptic exocytosis and endocytosis has been exploited in order to study the effects of protein phosphorylation on synaptic-vesicle cycling at the molecular level. The best-characterized protein in this respect is synapsin, whose function in the release of synaptic vesicles from the reserve pool is regulated by phosphorylation. More recently, it has emerged that proteins that function at other stages of the synaptic-vesicle cycle, which include priming of vesicles for docking-fusion and endocytic recycling, are also controlled by phosphorylation. Furthermore, recent work suggests that this regulation of membrane traffic by phosphorylation also occurs postsynaptically, where it contributes to synaptic plasticity.

Control of exocytosis in adrenal chromaffin cells

The major physiological stimulus for exocytosis in chromaffin cells is a rise in [Ca 2+ ] i . In this review I will discuss the nature of the Ca 2+ signal, the role of other second messengers and the factors that regulate or may be components of the Ca 2+ -dependent exocytotic mechanism

The Neuronal cytoskeleton

Cytoskeleton is a major neuronal organelle, R.Burgoyne molecular architecture and dynamics of the neuronal cyloskeleton, N.Hirokawa high molecular weight microtubule-associated proteins of the brain, R.Burgoyne properties and assembly of neuronal microtubules in vitro, R.Burns actin and actin-binding proteins in neurons, J.Bamburg and B.Bernstein neuronal plasma membrane associated cytoskeleton, A.Baines neurofilament proteins, G.Shaw structure and expression of neurofilament genes, J.P.Julien and F.Grosveld cytoskeleton of the growing axon, M.Cambray-Deakin microtubule-based organelle-transport of cytoskeletal proteins, R.Nixon cytoskeleton in secretion and neurotransmitter release, T.Cheek and R.Burgoyne.

Neuronal Ca2+-sensor proteins: multitalented regulators of neuronal function

Many aspects of neuronal activity are regulated by Ca2+ signals. The transduction of temporally and spatially distinct Ca2+ signals requires the action of Ca2+-sensor proteins including various EF-hand-containing Ca2+-binding proteins. The neuronal Ca2+ sensor (NCS) protein family and the related Ca2+-binding proteins (CaBPs) have begun to emerge as key players in neuronal function. Many of these proteins are expressed predominantly or only in neurons, sometimes with cell-specific patterns of expression. Their ability to associate with membranes either constitutively or in response to elevated Ca2+ concentration allows the NCS proteins to discriminate between different spatial and temporal patterns of Ca2+ signals. Recent work has established several physiological roles of these proteins, including diverse actions on gene expression, ion channel function, membrane traffic of ion channels and receptors, and the control of apoptosis.

Ca2+ and secretory-vesicle dynamics

Exocytosis in neurones and neuroendocrine cells is triggered by an increase in the cytosolic concentration of Ca2+, and is followed by endocytotic membrane retrieval. Electrophysiological studies have characterized the nature of the Ca2+ signal that is required for exocytosis, and have defined the Ca(2+)-dependent steps in exocytotic and endocytotic vesicle cycling. In parallel, biochemical approaches have led to the discovery of a range of proteins that appears to function in synaptic- and secretory-vesicle dynamics. The nature of the Ca(2+)-binding proteins, and how they interact with the identified components of the exocytotic and endocytotic machinery, remain key unresolved issues. However, it is apparent that exocytosis involves multiple Ca(2+)-binding proteins with different affinities, and that the Ca2+ sensor involved in the final membrane-fusion step has different affinities for Ca2+ in synapses and neuroendocrine cells.