Lipid bilayer dynamics in plasma and coated vesicle membranes from bovine adrenal cortex. Evidence of two types of coated vesicle involved in the LDL receptor traffic

Pure coated vesicles have been prepared from the bovine adrenal cortex and two homogeneous populations have been separated, one of large diameter (100 nm) and one of small diameter (70 nm). The chemical composition in lipids and proteins of coated vesicles has been compared with that of partially purified plasma membranes and evidences a higher protein/lipid ratio and a higher concentration in phosphatidylethanolamine and unsaturated fatty acids. Evaluation of the lateral diffusion of pyrene in the lipid bilayer of coated vesicles as compared to uncoated vesicles evidences a slowing-down effect of clathrin. Measurements of lipids' rotational diffusion by time-resolved fluorescence indicate a decrease in the order parameter of the lipids in the coated vesicles due to clathrin. A hypothesis is proposed for a possible role of the clathrin coat in the concerted motion of lipids and proteins toward coated pits and in the mechanism of formation of coated vesicles. Separation of the large from the small coated vesicles made it possible to reveal different protein components in the two types of vesicle by electrophoresis and autoradiograms of the [γ-³²P]adenosine triphosphate- (ATP-) treated vesicles. Visualisation of the low-density lipoprotein receptor by ligand blotting and enzyme-linked immunosorbent assay (ELISA) techniques indicates an increased low-density lipoprotein receptor binding capacity in small coated vesicles as compared to large ones and plasma membranes.     

Synaptic vesicle dynamics sans dynamin

The neuron-specific guanosine triphosphatase dynamin 1 has been hypothesized to be critically required for pinching off synaptic vesicles during endocytosis. In a recent publication in Science, Ferguson et al. describe a series of experiments demonstrating an unexpectedly selective requirement of dynamin 1 in synaptic vesicle endocytosis only during high frequency (<10 Hz) stimulation, but not after cessation of the stimulus train.     

Role of vesicle tethering factors in the ER-Golgi membrane traffic

Tethers are a diverse group of loosely related proteins and protein complexes grouped into three families based on structural and functional similarities. A well-accepted role for tethering factors is the initial attachment of transport carriers to acceptor membranes prior to fusion. However, accumulating evidence indicates that tethers are more than static bridges. Tethers have been shown to interact with components of the fusion machinery and with components involved in vesicle formation. Tethers belonging to the three families act at the same stage of traffic, suggesting that they mediate distinct events during vesicle tethering. Thus, multiple tether-facilitated events are required to provide selectivity to vesicle fusion. In this review, we highlight findings that support this model.     

Synaptic vesicle pools and dynamics

Synaptic vesicles release neurotransmitter at chemical synapses, thus initiating the flow of information in neural networks. To achieve this, vesicles undergo a dynamic cycle of fusion and retrieval to maintain the structural and functional integrity of the presynaptic terminals in which they reside. Moreover, compelling evidence indicates these vesicles differ in their availability for release and mobilization in response to stimuli, prompting classification into at least three different functional pools. Ongoing studies of the molecular and cellular bases for this heterogeneity attempt to link structure to physiology and clarify how regulation of vesicle pools influences synaptic strength and presynaptic plasticity. We discuss prevailing perspectives on vesicle pools, the role they play in shaping synaptic transmission, and the open questions that challenge current understanding.     

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes

It is well known that disrupted brain iron homeostasis was involved in Parkinson’s disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 μM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.     

Probing vesicle dynamics in single hippocampal synapses

We use fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study vesicle dynamics inside the synapses of cultured hippocampal neurons labeled with the fluorescent vesicle marker FM 1-43. These studies show that when the cell is electrically at rest, only a small population of vesicles is mobile, taking seconds to traverse the synapse. Applying the phosphatase inhibitor okadaic acid causes vesicles to diffuse freely, moving 30 times faster than vesicles in control synapses. These results suggest that vesicles move sluggishly due to binding to elements of the synaptic cytomatrix and that this binding is altered by phosphorylation. Motivated by these results, a model is constructed consisting of diffusing vesicles that bind reversibly to the cytomatrix. This stick-and-diffuse model accounts for the fluorescence correlation spectroscopy and fluorescence recovery after photobleaching data, and also predicts the well-known exponential refilling of the readily releasable pool. Our measurements suggest that the movement of vesicles to the active zone is the rate-limiting step in this process.     

Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.     

Spatiotemporal dynamics of the COPI vesicle machinery

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm² s⁻¹), whereas coatomer diffuses 5–10 times more slowly than expected (D ≈ 1 μm² s⁻¹). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.     

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of beta-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of beta-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.     

Computational model for autophagic vesicle dynamics in single cells

Macroautophagy (autophagy) is a cellular recycling program essential for homeostasis and survival during cytotoxic stress. This process, which has an emerging role in disease etiology and treatment, is executed in four stages through the coordinated action of more than 30 proteins. An effective strategy for studying complicated cellular processes, such as autophagy, involves the construction and analysis of mathematical or computational models. When developed and refined from experimental knowledge, these models can be used to interrogate signaling pathways, formulate novel hypotheses about systems, and make predictions about cell signaling changes induced by specific interventions. Here, we present the development of a computational model describing autophagic vesicle dynamics in a mammalian system. We used time-resolved, live-cell microscopy to measure the synthesis and turnover of autophagic vesicles in single cells. The stochastically simulated model was consistent with data acquired during conditions of both basal and chemically-induced autophagy. The model was tested by genetic modulation of autophagic machinery and found to accurately predict vesicle dynamics observed experimentally. Furthermore, the model generated an unforeseen prediction about vesicle size that is consistent with both published findings and our experimental observations. Taken together, this model is accurate and useful and can serve as the foundation for future efforts aimed at quantitative characterization of autophagy.     

Molecular dynamics simulations of lipid vesicle fusion in atomic detail

The fusion of a membrane-bounded vesicle with a target membrane is a key step in intracellular trafficking, exocytosis, and drug delivery. Molecular dynamics simulations have been used to study the fusion of small unilamellar vesicles composed of a dipalmitoyl-phosphatidylcholine (DPPC)/palmitic acid 1:2 mixture in atomic detail. The simulations were performed at 350-370 K and mimicked the temperature- and pH-induced fusion of DPPC/palmitic acid vesicles from experiments by others. To make the calculations computationally feasible, a vesicle simulated at periodic boundary conditions was fused with its periodic image. Starting from a preformed stalk between the outer leaflets of the vesicle and its periodic image, a hemifused state formed within 2 ns. In one out of six simulations, a transient pore formed close to the stalk, resulting in the mixing of DPPC lipids between the outer and the inner leaflet. The hemifused state was (meta)stable on a timescale of up to 11 ns. Forcing a single lipid into the interior of the hemifusion diaphragm induced the formation and expansion of a fusion pore on a nanosecond timescale. This work opens the perspective to study a wide variety of mesoscopic biological processes in atomic detail.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.