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Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

University of British Columbia

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (alpha(2)-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the alpha(7B) splice variant of the alpha(7) integrin receptor subunit to be up-regulated. These results support the possibility that alpha(7B) integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.

Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes.

Inwardly rectifying potassium (Kir) channels in Müller glia play a critical role in the spatial buffering of potassium ions that accumulate during retinal activity. To this end, Kir channels show a polarized subcellular distribution with the predominant channel subunit in Müller glia, Kir4.1, clustered in the endfeet of these cells at the inner limiting membrane. However, the molecular mechanisms underlying their distribution have yet to be identified. Here, we show that laminin, agrin and alpha-dystroglycan (DG) codistribute with Kir4.1 at the inner limiting membrane in the retina and that laminin-1 induces the clustering of alpha-DG, syntrophin and Kir4.1 in Müller cell cultures. In addition, we found that alpha-DG clusters were enriched for agrin and sought to investigate the role of agrin in their formation using recombinant C-agrins. Both C-agrin 4,8 and C-agrin 0,0 failed to induce alpha-DG clustering and neither of them potentiated the alpha-DG clustering induced by laminin-1. Finally, our data reveal that deletion of the PDZ-ligand domain of Kir4.1 prevents their laminin-induced clustering. These findings indicate that both laminin-1 and alpha-DG are involved in the distribution of Kir4.1 to specific Müller cell membrane domains and that this process occurs via a PDZ-domain-mediated interaction. Thus, in the basal lamina laminin is an essential regulator involved in clearing excess potassium released during neuronal activity, thereby contributing to the maintenance of normal synaptic transmission in the retina.

Dystroglycan and Kir4.1 coclustering in retinal Müller glia is regulated by laminin-1 and requires the PDZ-ligand domain of Kir4.1.

In a previous study we showed that juvenile rats exposed, for various durations of time, to a bright luminous environment between P14 (eye opening) and P34 developed a light-induced retinopathy (LIR), the severity of which depending on the duration of exposure as well as the age of the rat at the onset of exposure. Our study also revealed that the severity of the LIR increased as the time elapsed between the cessation of exposure and the structural/functional evaluation increased, suggesting that the LIR degenerative process proceeded in two distinct steps namely, an initial (rapid) acute phase that was followed by a (slower) chronic phase. In view of the above, the purpose of the present study was to reinvestigate previous claims suggesting that exposure to bright light prior to eyelid opening had no measurable consequences on the retinal structure and function; the claim being that despite a non-detectable acute phase, bright light exposure prior to eyelid opening could nonetheless yield a significant retinopathy during the chronic phase of development of LIR. In order to test our hypothesis, neonatal rats were raised in a bright luminous environment from birth to P14. At P30, analysis of the results obtained from rats exposed between P0-P14 did not reveal, as previously acknowledged by others, significant LIR damages. However, results obtained at P60 disclosed significant functional anomalies with relative sparing of the retinal ultrastructure. Our results confirm that, in spite of closed eyelids, postnatal exposure to bright environment did trigger a slow degenerative process.

Structural and functional consequences of bright light exposure on the retina of neonatal rats.

The dystroglycan protein complex provides a link between the cytoskeleton and the extracellular matrix (ECM). Defective O-glycosylation of alpha-dystroglycan (alpha-DG) severs this link leading to muscular dystrophies named dystroglycanopathies. These are characterized not only by muscle degeneration, but also by brain and ocular defects. In brain and retina, alpha-DG and ECM molecules are enriched around blood vessels where they may be involved in localizing the inwardly rectifying potassium channel, Kir4.1, and aquaporin channel, AQP4, to astrocytic endfeet. To investigate in vivo the role of ECM ligand-binding to glycosylated sites on alpha-DG in the polarized distribution of these channels, we used the Large(myd) mouse, an animal model for dystroglycanopathies. We found that Kir4.1 and AQP4 are lost from astrocytic endfeet in brain whereas significant labeling for these channels is detected at similar cell domains in retina. Furthermore, while both alpha- and beta1-syntrophins are lost from perivascular astrocytes in brain, labeling for beta1-syntrophin is found in retina of the Large(myd) mouse. These findings show that while ligand-binding to the highly glycosylated isoform of alpha-DG in concert with alpha- and beta1-syntrophins is crucial for the polarized distribution of Kir4.1 and AQP4 to functional domains in brain, distinct mechanisms may contribute to their localization in retina.

Distribution of potassium ion and water permeable channels at perivascular glia in brain and retina of the Large(myd) mouse.

mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK), and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades.

Localized Rho GTPase activation regulates RNA dynamics and compartmentalization in tumor cell protrusions.

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.

Several recent studies have shown that neuroligin 2 (NL2), a component of the cell adhesion neurexins-neuroligins complex, is localized postsynaptically at hippocampal and other inhibitory synapses throughout the brain. Other studies have shown that components of the dystroglycan complex are also localized at a subset of inhibitory synapses and are coexpressed with NL2 in brain. These data prompted us to undertake a comparative study between the localization of NL2 and the dystroglycan complex in the rodent retina. First, we determined that NL2 mRNA is expressed both in the inner and in the outer nuclear layers. Second, we found that NL2 is localized both in the inner and in the outer synaptic plexiform layers. In the latter, the horseshoe-shaped pattern of NL2 and its extensive colocalization with RIM2, a component of the presynaptic active zone at ribbon synapses, argue that NL2 is localized presynaptically at photoreceptor terminals. Third, comparison of NL2 and the dystroglycan complex distribution patterns reveals that, despite their coexpression in the outer plexiform layer, they are spatially segregated within distinct domains of the photoreceptor terminals, where NL2 is selectively associated with the active zone and the dystroglycan complex is distally distributed in the lateral regions. Finally, we report that the dystroglycan deficiency in the mdx(3cv) mouse does not alter NL2 localization in the outer plexiform layer. These data show that the NL2- and dystroglycan-containing complexes are differentially localized in the presynaptic photoreceptor terminals and suggest that they may serve distinct functions in retina.

Synaptic localization of neuroligin 2 in the rodent retina: comparative study with the dystroglycan-containing complex.

Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clustered at the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role of the dystroglycan complex and its interaction with perivascular laminin in the clustering of AQP4 at perivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema.

A high throughput screen identifies chemical modulators of the laminin-induced clustering of dystroglycan and aquaporin-4 in primary astrocytes.

The proper targeting of ion channels to specialized domains is crucial for cell function. Kir4.1, the inwardly rectifying potassium channel, and aquaporin-4 (AQP4), the type 4 water-permeable channel, are localized at the basolateral domain of polarized epithelial cells; however, the mechanisms involved in their localization have yet to be determined. In this study, we investigated the role of the extracellular matrix in the localization of these channels in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells expressing green fluorescent protein-Kir4.1 or -AQP4 were cultured on laminin-1 or fibronectin and examined by confocal microscopy and cell surface biotinylation to assess plasma membrane expression of Kir4.1 and AQP4. Our data show that laminin-1 and fibronectin induce a significant increase in cell surface expression of both channels at the basolateral domain. Using fluorescence recovery after photobleaching, we demonstrate that laminin-1 and fibronectin reduce the diffusion rates of these channels. Finally, we show that the laminin receptor dystroglycan is important for cell surface expression of Kir4.1 but not AQP4. However, laminin-1 increases cell surface expression of both channels in cells deficient for dystroglycan, indicating that other receptors are involved. Indeed, RGD-containing peptides, which inhibit fibronectin binding to certain integrins, prevent the fibronectin-induced increase in Kir4.1 and AQP4 cell surface expression and reverse the laminin- and fibronectin-induced reduction in both channels' diffusion rates. Similarly, the αvβ3-integrin function-blocking antibody alters the reduction of AQP4 diffusion rates induced by both laminin and fibronectin, suggesting that αvβ3-integrin plays a role in the stabilization of APQ4 at the basolateral domain of epithelial cells.

Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix.

The water-permeable channel aquaporin-4 (AQP4) is highly expressed in perivascular astrocytes of the mammalian brain and represents the major conduit for water across the blood-brain barrier. Within these cells, AQP4 is found in great quantities at perivascular endfoot sites but is detected in lesser amounts at the membrane domains within the brain parenchyma. We had previously established that this polarization was regulated by the interaction between dystroglycan (DG), an extracellular matrix receptor that is co-expressed with AQP4, and the laminin that is contained within the perivascular basal lamina. In the present study, we have attempted to describe the mechanisms that underlie this regulation, using primary astrocyte cultures. Via biotinylation, we found that the cell-surface expression of AQP4 is DG-dependent and is potentiated by laminin. We also determined that this laminin-dependent increase occurs not through an upregulation of total AQP4 levels, but rather from a redirection of AQP4 from an intracellular, EEA-1-associated pool to the cell surface. We then demonstrated an association between DG and dynamin and showed that dynamin functioned in conjunction with clathrin to regulate surface AQP4 amounts. Furthermore, we observed that DG preferentially binds to the inactive forms of dynamin, suggesting that this interaction was inhibitory for AQP4 endocytosis. Finally, we showed that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4. Our data therefore indicate that the dual interation of DG with laminin and dynamin is involved in the regulation of AQP4 internalization, leading to its asymmetric enrichment at perivascular astrocyte endfeet.

Hakima Moukhles

Department of Cellular and Physiological Sciences

University of British Columbia

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Hakima Moukhles

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cellular and Physiological Sciences

University of British Columbia

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Department of Cellular and Physiological Sciences