Lipid membrane nanodomains are membrane areas enriched on proteins that can form oligomers and cluster in the membranes. The formation of these oligomers is favored by cholesterol and other lipid species. The described size of these domains is diverse, from 10–200 nm diameter, and their characteristics are sometimes associated with the lipid microenvironment ruling the interaction between cholesterol- and sphingolipids and proteins enriched in these domains, gathering different proteins with different roles, in the same domain. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain.
1. Lipid Membrane Nanodomains Organization in the Neuronal Plasma Membrane
The classical model of the plasma membrane, named the fluid mosaic model, described by Jonathan Singer and Garth Nicolson in 1972, is excessively reductionist for properly accounting for the well-organized plasma membrane domains. Lipid rafts are plasma membrane large areas of 10 and 200 nm diameter in size enriched in cholesterol and sphingolipids
[1]. The existence lipid rafts was initially a subject of debate between physical chemists and histologists due to difficulties in visualizing them and their ill-defined molecular composition
[1][2]. In the last two decades, a number of new techniques such as single-molecule spectroscopy, super-resolution microscopy, fluorescence recovery after photobleaching, stimulated emission depletion, Förster resonance energy transfer (FRET), total internal reflection fluorescence, and fluorescence correlation spectroscopy techniques allowed to estimate the lower limit of lipid rafts in <20 nm
[3][4][5][6]. Plasma membrane domains of 26 ± 13 nm radius have been observed in living cells diffusing as one entity for minutes
[7]. Further work using stimulated emission depletion (STED) far-field fluorescence nanoscopy revealed spots sized 70-fold below the diffraction barrier transiently trapped between 10 and 20 ms, in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas
[3]. The diffraction limit of visible light impedes domains smaller than 1 µm to be directly visualized and indeed large micrometer-sized lipid rafts domains are readily observed in artificial membranes
[3]. Also, associated proteins can mask the direct observation of lipid rafts in living cells. A tentative attempt to determine analogous domains in living cells has been made based on homo-FRET efficiencies obtained through the rate of fluorescence anisotropy loss and using GFP labeled glycosyl-phosphatidylinositol-anchored proteins which allow an estimation of the upper size limit of lipid rafts at ~5 nm
[8][9]. Yethiraj and Weisshaar have suggested that the spatial heterogeneity in cell membranes limits the transferability of the conclusion drawn from artificial membranes to live cells, as integral membrane proteins attached to the cytoskeleton act as obstacles that limit the size of lipid domains
[8].
For all these reasons, the reswearchers introduce the concept of lipid membrane domains in this researchview, arising from the fact that some membrane proteins form oligomers and clusters in the membranes, which formation is favored by cholesterol and other lipid species.
Regarding the protein components associated with lipid membrane domains, widely named in the bibliography as lipid rafts, a proteomic study identified up to 36 integral membrane proteins associated with lipid membrane domain and flotillin, as a marker of these membrane domains where identified in the human brain
[10]. In another study, 175 membrane-associated proteins were identified by proteomics, including L-type calcium channels and the plasma membrane calcium ATPase (PMCA), using caveolin-1 (Cav-1) and flotillin-1 (Flot-1), as biomarkers of lipid membrane domains isolated from brain neonatal mice
[11]. Similarly, a proteomic assessment of proteins present in isolated lipid membrane domains of adult mouse brains identified 133 proteins, using Flot-1 as a marker of plasma membrane domains
[12]. This study also highlighted the colocalization of this protein with several calcium channel subunits
[12]. In cultured hippocampal neurons, sphingolipid-cholesterol-enriched microdomains have been localized flotillin 1, Thy-1 cell surface antigen or CD90, as specific lipid membrane-domain markers, associated with the ganglioside named monosialotetrahexosylganglioside (GM1)
[13]. It is worth to mention at this point that although GM1 is not a definite lipid membrane-domain marker, its distribution into lipid membrane domains depends on the concentration. At elevated concentration, GM1 can form its own domains organizing in the plasma membrane in non-lipid membrane-domain areas located predominantly in the L
d phase
[14]. Very recent discoveries regarding the molecular architecture of lipid membrane nanodomains support their organization in planar tightly packed nanodisks of Cav-1, with a 140Å external diameter size
[15]. It is also probable that a similar size supramolecular complex based on flotillin might exist, based on the observed structural conformations of stomatin, prohibitin, flotillin, and the modulator for HflB protease specific for phage lambda cII repressor (HflK/C) domains (SPFH domain)
[16]. Also, some studies have reported the isolation of up to 4 types of domains in the plasma membrane at physiological conditions
[17]. Given the existence of these nanostructures, a question arises regarding how many of the reported protein molecules in the aforementioned proteomic and non-proteomic studies
[10][11][12][13] could fit within a single of these nanostructures on one neuronal lipid membrane domain. The quantity of proteins reported in the neuronal lipid membrane domain contrasts with the number of proteins that could fit within or surrounding a 140Å diameter size nanodisk, if this type of structure stands alone as the main component of neuronal planar-lipid membrane domains in the plasma membrane. Neuronal lipid membrane domains are different from those of the invaginated
caveolae in a variety of cell types, which require the presence of the protein named cavin and higher-order interactions with other proteins
[18][19][20]. Cav-1–cavin interaction seems required to form mature
caveolae, which have a polygonal shape to induce curvature in non-neuronal cells
[21][22][23][24]. Cavin is absent or released when conforming planar-non-invaginated lipid membrane domains
[20][25][26][27], like those described in neuronal lipid membrane domains.
In addition to these studies, more efforts are required to ascertain whether Cav-1 nanodisks independently exist in neuronal cells, either as discrete entities supporting non-invaginated areas on the plasma membrane or as components of supramolecular structures analogous to those observed in invaginated
caveolae [20]. Since supramolecular structures with a similar protein composition to that of
caveolae do not exist in neurons, the presence of a high number of proteins located in lipid membrane domains raises questions regarding the number of proteins that one Cav-1 nanodisk can hold due to steric hindrance. Methods for lipid membrane-domain isolation based on differential gradient centrifugation cannot discern the existence of lipid membrane nanodomain subtypes. Particularly, cytochemical and histochemical studies combined with physicochemical techniques based on quantitative fluorescence energy transfer (FRET) techniques, as those conducted by the research group led by Prof. Gutierrez-Merino, have provided insights into this matter by identification of proteins in clusters complexing with protein markers of lipid membrane domains (caveolin and flotillin isoforms) at a distance <100 nm in studies performed in neurons and brain tissue using the appropriate secondary fluorescent antibodies against the primary antibody of the selected lipid membrane-domain marker
[28][29][30][31][32][33][34][35][36]. As discussed in these articles, this is a particular case of FRET from one donor to multiple acceptors, a situation in which the maximum range of FRET distance is significantly expanded, as analyzed in detail in former studies with purified biological membranes
[23][37][38][39]. These research findings might support the existence of clusters that could stand alone as individual entities, such as Cav-1 nanodisks, with a diverse variety of calcium transporter elements. The well-recognized and wide distribution of these transporters in neurons, functioning as partners of lipid membrane-domain markers, strongly suggests the potential existence of multiple lipid membrane-domain subtypes within neurons. A neuronal lipid membrane-domain subtype is defined in this work as a plasma membrane, synaptic or extrasynaptic structure characterized by the presence of a protein biomarker of lipid membrane nanodomain and a specific calcium transport systems. The existence of these subdomains might correlate with the function of calcium gradients associated with cytosolic calcium microcompartments, near the plasma membrane
[33][40], and such patterns may arise under certain conditions
[41][42][43][44].
In this context, it is intriguing and controversial whether different types of lipid membrane domains might exist within a single cell or across different cell types based on the complex lipid and protein composition of these domains. This issue might be particularly notorious in tissues such as the brain, where recent findings using single-cell sequencing and methods to map the spatial location of gene expression have unraveled the extraordinary cellular diversity existing within this tissue
[45]. Strategies for isolating lipid membrane domains, named rafts in these studies, that utilized membrane tension generate large observable membrane domains or lipid rafts, that are converted into small ones when the tension was relieved
[17]. This result lends support to the hypothesis that a myriad of not well-described plasma membrane nanodomains might exist.
For cells, application of membrane tension resulted in several types of large domains; one class of domains was identified as a lipid raft, defined here as lipid membrane domain. Furthermore, the distribution of protein components of lipid domains
[46][47][48][49] in planar non-invaginated regions of the neuronal plasma membrane
[20][25][26][27], may be considered a robust evidence for the existence of not-so-transient, underlying structures that support several membrane nanodomains in neurons. This structural arrangement may differ from that observed in other cell types, where membrane invaginated areas forming
caveolae have been described involved in membrane trafficking, with a transient formation and elimination of the protein content of these domains.
2. Properties of Caveolin-, Flotillin- or Ganglioside-Containing Lipid Membrane Domains
Within neuronal lipid membrane domains, at least two classes of protein, named caveolin and flotillin, can scaffold cholesterol and have been used as biomarkers of these domains
[50][51][52][53][54][55][56]. The differential spatial distribution of the caveolin-, flotillin- or some specific lipid-enriched domains of the neuronal plasma membrane suggests that various domains co-exist in one neuron. They will be called caveolin- and flotillin-enriched lipid membrane domains. Their differential association with plasma membrane receptors acting through calcium signaling, as well as with calcium channels and transport systems might be useful to classify lipid membrane nanodomains. Other lipids, such as gangliosides have been associated with both in certain contexts but not always
[57][58][59]. This supports the idea that their presence might constitute a marker for additional lipid membrane nanodomain subtypes. The characterization and differentiation between these domains have been challenged by the limitations and insufficient resolution of the conventional methods for preparative isolation of lipid domains using a whole brain tissue or cells in culture (
Figure 1). This is a major handicap for a proper classification of lipid membrane-domain subtypes. A potential dissection through immunohistochemical and immunocytochemical methods could offer insights of their precise intracellular and intercellular locations. Moreover, this dissection could contribute to a better comprehension of how key plasma membrane components in charge of calcium homeostasis are regulated in lipid membrane domains.
Figure 1.
Some of the potential lipid membrane domains that can be isolated from brain tissue are associated with different neuronal types.
3. The Summary of the Distribution Map
A wide range of possible complexes enriched in lipid membrane nanodomain subtypes in the same or different glutamatergic neurons has been described. The organization of NMDAR, L-P/Q calcium channels, some metabotropic receptors, and PMCA located in the synapses of glutamatergic neurons are shown in
Figure 2.
Figure 2. Illustration of a variety of caveolin- and flotillin-enriched lipid membrane domains location complexing with calcium transporter elements (NMDAR, L-P/Q calcium channels, some metabotropic receptors, and PMCA) in synaptic terminals described to exist in glutamatergic neuronal cells. Calcium transporter elements have been differentially described to be present in many neuronal locations, including somas, neurites, axons, dendrites, spines, and synaptic terminals. In synaptic terminals a variability of subunits may yield specific calcium transporters for that location (i.e.,: presynaptic and postsynaptic NMDAR might be differentiated by the type of subunits that configure them in hippocampal neurons
[60]) that might differ in configuration from those distributed in other neuronal locations and vary in respect to the neuronal cell type
[61]. In this figure,
thwe
researchers are are focusing on calcium-transporting elements associated with caveolin- and flotillin-enriched lipid membrane domains, that should be added to those elements that are not located in lipid membrane-domain areas (not shown in this figure) and omitted in synaptic terminals that comprise areas of 0.5 to 2 μm size
[62]. Lipid membrane domains associated with gangliosides are suggested to be involved in endocytic processes in some membranes and have been omitted from this figure for the sake of clarity. NMDA (1) and L-type calcium channels (2) located in caveolin-enriched domains might function as redox nanotransducers in charge of the control of these calcium transporters working as a microchip-like structure for a tighter functional coupling between calcium, nitric oxide and superoxide anion signaling in presynapses
[32][63][64] and postsynapses (3) also sensitive to superoxide anion
[65][66], in glutamatergic CGNs. Also, in associated caveolin-enriched domains at presynapses, we can allocate PMCA (4), which are susceptible of inhibition by GM1 contained in these subdomains
[33][67][68]. PMCA has also been described to be present in flotillin-enriched lipid membrane domains (5), which are very sensitive to cholesterol content. The activity in these domains is higher than the one not present in lipid membrane domains
[69]. The differential response to endogenous cholesterol and gangliosides seems to support that caveolin and flotillin-enriches domains constitute different lipid membrane nanodomain subtypes in presynaptic terminals. We can also find P/Q-type calcium channels at presynaptic terminals (6) associated with flotillin-enriched domains and GPI-enriched areas
[70][71][72]. This type of cluster can also be found in postsynaptic terminals (7)
[73], and the physiological behavior has been characterized by the presence of Flot-1 and has been related to and increases in the frequency of miniature excitatory postsynaptic currents. Several subunits of metabotropic receptors have been described to colocalize in caveolin- and flotillin-enriched domains (8) and (9). Subunit interaction with caveolin has been better described than for flotillin. Several motifs of mGlu subunits have been described to interact with cav-1
[74][75], which controls the rate of receptor internalization and location at the surface
[74][75]. A function of recruitment of NMDARs into lipid membrane domains at postsynapses to initiate second messenger signaling cascades linked with receptor depletion for neuronal protection in NMDAR-induced excitotoxicity has been suggested for NMDAR located at flotillin-enriched domains (10)
[76]. As previously indicated, NMDARs can associate with scaffold protein PSD-95 and form signaling complexes that differ in their composition. Some subunits of the AMPAR have also been located in caveolin- (11) and flotillin-enriched domains (12) at post synaptic terminals associated with PSD-95. NO
• has a similar effect mimicking that of NMDA, recruiting AMPARs to lipid membrane-domain surface which suggest a counterplay with lipid membrane domains associated with postsynaptic domains (3) or presynaptic (1) and (2) domains since NO
• can reach this location by diffusion from presynaptic sources.
A summary of the components implicated in calcium signaling in neurons and their association and function with each lipid membrane-domain subtype can be found in the
Table 1.
Table 1.
Calcium signaling components and distribution map in lipid raft-domain subtypes.
Type
|
Subunit
|
Neuronal Type
|
Associated with Raft Component
|
Main Distribution in Brain and Subcellular Location
|
Function
|
L-type
|
Cav1.2
|
Primary culture of cerebellar granule neurons and Purkinje cells [30][77]
|
Cav-1 and GM1 [30], GM1 [77]
|
|
Neuronal calcium transients in cell bodies and dendrites, regulation of enzyme activity, regulation of transcription [78]
|
P/Q-type
|
Cav2.1
|
Cerebellar Purkinje neurons (tissue [70]; primary culture [79]; brain synaptosomal fraction [71])
|
Flot-1 [70], GM1 [71][79]
|
Hippocampus [80], dorsal root ganglion neurons [81], presynaptic areas [71][81]
|
Neurotransmitter release, dendritic calcium transients [78]
|
L/P/Q/N-type
|
α2δ-2, α2δ-3 [72]
|
Hippocampal neurons (raft isolation and microscopy) [72]
|
Flot-1 [72]
|
GPI-enriched areas [72]
|
|
NMDA
|
NR1
|
Primary cultures of hippocampal neurons [73]; ganglion cells in rat retina (tissue) [82][83]; ventral part of lamina III and in laminae III and IV [84]
|
Flot-1 [73]; GM1 [82][83][84]
|
Small uniform puncta throughout the neuron, pre and postsynapse [73][84]; ganglion cell dendrites [82], extrasynaptic plasma membrane [83]
|
Signaling complexes in the postsynaptic density [85], glutamatergic signaling, synaptic plasticity, excitotoxicity, and memory [65], neurite outgrowth and axonal growth cone motility [86][87]
|
NR2B
|
Anterior cingulate cortex neurons in tissue and cultured (microscopy and immunoprecipitation) [88]; neurons from normal rat cerebral cortex (raft isolation, microscopy and immunoprecipitation) [89]; primary culture of cortical neurons (microscopy and raft isolation) [65]; ganglion cells in rat retina (tissue) [82][83]
|
Cav-1 [88][89], Flot-1 [89]; GM1 [82][83]
|
Soma and postsynapses [88][89]; ganglion cell dendrites extrasynapses peri-synapses [82][83]
|
NR2A [90]
|
Cultured hippocampal neurons (microscopy and raft isolation) [90]
|
Flot-1 and -2 [90]
|
Small uniform puncta throughout the neuron [90]
|
AMPAR
|
GluA2 [91]
|
Primary culture of hippocampal neurons (microscopy, immunoprecipitation and raft preparation) [91]
|
Cav-1 [91],
|
Cell body and as puncta localized to areas of cellular outgrowth [91]
|
Postsynaptic currents mediated by the AMPA subtype of glutamate receptors in LTP [92]; long-term potentiation (LTP) induced GluA1 surface exposure [93]
|
GluA1 [94][95]
|
Primary culture of hippocampal neurons (microscopy and raft isolation) [94][95]
|
Flot-1 and -2 [95], Cav-1 [96],
GM1 [94]
|
Postsynapses [94], synapses and dendritic Spines [96]
|
GluR2/3 [96]
|
Primary culture of hippocampal neurons (microscopy) [96], synaptosomes [97]; ganglion cells in rat retina (tissue) [82]
|
Cav-1 [96], GM1 [82][97]
|
Synapses and dendritic spines [96]; dendrites and somata [82]
|
GluR4
|
Ganglion cells in rat retina (tissue) [82]
|
GM1 [82]
|
Dendrites and somata [82]
|
mGluR
|
mGluR1/5
|
Primary hippocampal neurons (microscopy and immunoprecipitation) [74]
|
Cav-1 [74]
|
Soma and dendrites [74]; postsynaptic density late in development [98]
|
Synapse formation and plasticity [75]
|
|
mGluR1a
|
Hippocampus, arcuate nucleus, hypothalamus [99]
|
Cav-1 [99]
|
|
Caveolin proteins act to functionally isolate distinct estrogen receptors and mGluRs, leading to activation of specific second messenger signaling cascades [99]
|
|
mGluR1α
|
Synaptosomes from pig cerebellum
|
Cav-1 and Flot [100][101]
|
|
By application of MβCD, interaction of phosphorylated caveolin with the receptor decreased, and finally, internalization of the receptor was blocked [100]
|
Pumps
|
PMCA isoform 4
|
Synaptosomes from pig cerebellum (Brij96 extracts) [102]
|
ganglioside GM1 [102]
|
|
Discrete functional positions on the synaptic nerve terminals [102]
|
Purinergic receptors
|
P2X3
|
Rat brain, cerebellar granule neurons in culture (microscopy, immunoprecipitation and raft preparation), dorsal root ganglion neurons in culture
|
Flot-2, Cav-1
|
P2X3 subunit is expressed in cell bodies as well as in peripheral and central terminals of sensory neurons in dorsal root ganglia (DRG) [103][104]
|
Well-defined role in pain perception [105][106]. Cav-1 is required for basal and ligand-induced membrane delivery of the P2X3 receptor [107]
|
Note: The reason for no data regarding some of the calcium components and the main distribution in brain and subcellular location is the description of these calcium components in experiments performed in vitro in culture. Although some of these cultures were prepared from tissue, the researchers thought this should be differentiated from histochemical studies reporting calcium transported elements in rafts directly visualized on tissue slices or directly prepared or isolated from those tissues.