Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains

Individual growth factors can regulate multiple aspects of behavior within a single cell during differentiation, with each signaling pathway controlled independently and also responsive to other receptors such as cell surface integrins. The mechanisms by which this is achieved remain poorly understood. Here we use myelin-forming oligodendrocytes and their precursors to examine the role of lipid rafts, cholesterol and sphingolipid-rich microdomains of the cell membrane implicated in cell signaling. In these cells, the growth factor PDGF has sequential and independent roles in proliferation and survival. We show that the oligodendrocyte PDGFalpha receptor becomes sequestered in a raft compartment at the developmental stage when PDGF ceases to promote proliferation, but is now required for survival. We also show that laminin-2, which is expressed on axons in the CNS and which provides a target-dependent signal for oligodendrocyte survival by amplification of PDGFalphaR signaling, induces clustering of the laminin binding integrin alpha6beta1 with the PDGFalphaR-containing lipid raft domains. This extracellular matrix-induced colocalization of integrin and growth factor receptor generates a signaling environment within the raft for survival-promoting PI3K/Akt activity. These results demonstrate novel signaling roles for lipid rafts that ensure the separation and amplification of growth factor signaling pathways during development.     

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.

To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.     

Membrane microdomains in immunoreceptor signaling

Membrane microdomains denoted commonly as lipid rafts (or membrane rafts) have been implicated in T-cell receptor (TCR), and more generally immunoreceptor, signaling for over 25 years. However, this area of research has been complicated by doubts about the real nature (and even existence) of these membrane entities, especially because of methodological problems connected with possible detergent artefacts. Recent progress in biophysical approaches and functional studies of raft resident proteins apparently clarified many controversial aspects in this area. At present, the prevailing view is that these membrane microdomains are indeed involved in many aspects of cell biology, including immunoreceptor signaling. Moreover, several other types of raft-like microdomains (perhaps better termed nanodomains) have been described, which apparently also play important biological roles.     

Calcium signalling in oligodendrocytes

Generation of calcium signal in mammalian oligodendrocytes is a result of two different mechanisms, namely: (i) transmembrane calcium influx via voltage-operated calcium channels, and (ii) calcium release from IP₃-sensitive internal calcium stores. The oligodendrocytes express two types of voltage-operated calcium channels with different voltage-dependence. The expression of calcium channels undergoes marked changes during the development of the oligodendrocytes. The cytoplasmic calcium release from internal depots can be triggered by the activation of P₂ metabotropic purinoreceptors.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 26–31, January–February, 1994.     

Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca²⁺ triggers exocytosis. The existence of sub-micrometer domains of greater than 100 μM [Ca²⁺]_i was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca²⁺ ‘microdomains’ in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca²⁺ nano- and microdomains forming around the mouth of voltage-gated Ca²⁺ channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca²⁺ channels at different central synapses and pointed out the role of Ca²⁺ syntillas – localized, store operated Ca²⁺ signals – in facilitation and spontaneous release. The coupling between Ca²⁺ increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca²⁺ comes from different sources, including Ca²⁺ influx via the plasma membrane and the mobilization of Ca²⁺ from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca²⁺ signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca²⁺ signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca²⁺ sources, leading to a wide spectrum of Ca²⁺ signals triggering release.     

Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca(2+) triggers exocytosis. The existence of sub-micrometer domains of greater than 100 microM [Ca(2+)](i) was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca(2+) 'microdomains' in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca(2+) nano- and microdomains forming around the mouth of voltage-gated Ca(2+) channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca(2+) channels at different central synapses and pointed out the role of Ca(2+) syntillas - localized, store operated Ca(2+) signals - in facilitation and spontaneous release. The coupling between Ca(2+) increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca(2+) comes from different sources, including Ca(2+) influx via the plasma membrane and the mobilization of Ca(2+) from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca(2+) signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca(2+) signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca(2+) sources, leading to a wide spectrum of Ca(2+) signals triggering release.     

Signaling microdomains in T cells

Sub-micron scale signaling domains induced in the plasma membrane of cells are thought to play important roles in signal transduction. In T cells, agonist MHC-peptide complexes induce small diffraction-limited domains enriched in T cell receptor (TCR) and signaling molecules. These microclusters serve as transient platforms for signal initiation and are required for sustained signaling in T cells, although each microcluster functions for only a couple of minutes. How they are formed, and what mechanisms promote and regulate signaling within TCR microclusters is largely unknown, although it is clear that TCR engagement and dynamic reorganization of cortical actin are involved. Here, we review current understanding of signaling within microclusters in T cells, and speculate on how these structures may form, initiate biochemical signals, and serve as sites of both signal integration and amplification, while also facilitating appropriate termination of TCR and related signaling.     

Calcium microdomains in aspiny dendrites

Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Plasma membrane microdomains

Several lines of evidence indicate that the lipids in the plasma membrane of animal cells are inhomogeneously distributed, and that various types of specialized lipid domains play an important role in many biological processes. The characteristics of these domains, such as size, composition and dynamics, are currently under active investigation. It appears that there are many different types of membrane domains in the plasma membrane, and perhaps the entire membrane should be viewed as a mosaic of microdomains.     

Imaging single-channel calcium microdomains

The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions.     

NMDA receptors expressed in oligodendrocytes

Oligodendrocytes are known to express (Ca2+)-permeable glutamate receptors and to have low resistance to oxidative stress, two factors that make them potentially susceptible to injury. Oligodendrocyte injury is intrinsic to the loss of function experienced in conditions ranging from cerebral palsy to spinal cord injury, focal ischaemia and multiple sclerosis. NMDA receptors, a subtype of glutamate receptors, are vital to the remodeling of synaptic connections during postnatal development and associative learning abilities in adults and possibly in improvements in oligodendrocyte function. Previous studies had failed to detect NMDA receptor mRNA or current in oligodendrocytes but three new papers demonstrate NMDA receptor expression in oligodendrocytes and discuss its implications for ischaemia therapy.     

Calcium microdomains in mitochondria and nucleus

Endomembranes modify the progression of the cytosolic Ca(2+) wave and contribute to generate Ca(2+) microdomains, both in the cytosol and inside the own organella. The concentration of Ca(2+) in the cytosol ([Ca(2+)](C)), the mitochondria ([Ca(2+)](M)) and the nucleus ([Ca(2+)](N)) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca(2+) from the high [Ca(2+)](C) microdomains generated during cell activation near Ca(2+) channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca(2+) signal to the bulk cytosol. This shaping of [Ca(2+)](C) signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca(2+)](M) signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca(2+)](C) signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca(2+)](N) signals could be generated by direct release of stored Ca(2+) into the nucleoplasm. Ca(2+) release could even be restricted to subnuclear domains. Putative Ca(2+) stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca(2+) into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain.     

Glutamate receptor dynamics in dendritic microdomains

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.     

Imaging single-channel calcium microdomains

The Ca²⁺ microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca²⁺ signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca²⁺ imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of ‘optical patch-clamp recording’ is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca²⁺ ions.     

Presence of detergent-resistant microdomains in lysosomal membranes

We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.     

Tight junctions of oligodendrocytes

Summary Freeze-fracture replicas of the rat corpus callosum revealed prominent junctional strands in fractured cell membranes of the somata of oligodendrocytes. The junctional strands were characterized by an elaborate system of straight or slightly undulating rows of linear aggregates of particles or ridges in the P face and furrows in the E face.     

Signaling in dendritic spines and spine microdomains

The specialized morphology of dendritic spines creates an isolated compartment that allows for localized biochemical signaling. Recent studies have revealed complexity in the function of the spine head as a signaling domain and indicate that (1) the spine is functionally subdivided into multiple independent microdomains and (2) not all biochemical signals are equally compartmentalized within the spine. Here we review these findings as well as the developments in fluorescence microscopy that are making possible direct monitoring of signaling within spines and, in the future, within sub-spine microdomains.     

Ca2+ microdomains in smooth muscle

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.