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Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes.

Smith AJ, Jin BJ, Ratelade J, Verkman AS - J. Cell Biol. (2014)

Bottom Line: The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays.Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix.Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, CA 94143.

ABSTRACT
The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1-AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23-AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

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ECM adhesion and polarization to astrocyte endfeet requires M23–AQP4. (A) Wide-field and TIRF images of U87-MG cells stably transfected with M1– or M23–AQP4 and stained with antibody to AQP4 and for F-actin with phalloidin. Top panels shows untreated control cells; in bottom panels live cells were subject to detergent extraction by treatment with 0.5% Triton X-100 for 5 min before fixation. (B) Mean cellular AQP4 immunofluorescence staining intensity measured from TIRF images of M1- or M23-transfected cells under control conditions or with 0.5% Triton X-100 treatment before fixation. (n = 5 cells each, ±SE; n.s., P > 0.05; ***, P < 0.001 by t test). (C) TIRF images and ratio image of live U87-MG cells transfected with M1–AQP4–GFP and M23–AQP4–mCherry before and after incubation with 0.5% Triton X-100. (D) Quantification of M1/M23 ratio in transfected U87-MG cells before and after incubation with 0.5% Triton X-100; *, P < 0.05 by paired t test. (E) AQP4 distribution in astrocytes after intracerebral injection of M1– (top) or M23–AQP4 (bottom) adenoviruses into AQP4−/− mice. Left panels show AQP4 distribution in GFAP-labeled astrocytes adjacent to cerebral blood vessels (dotted line), right panels show an enlarged view of AQP4 and GFAP staining in astrocyte end-feet (labeled “E”). (F) Quantification of the ratio of AQP4 staining density in GFAP-labeled processes adjacent to blood vessels versus nonadjacent processes for cells transfected with M1– or M23–AQP4; **, P < 0.01 by t test.
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fig7: ECM adhesion and polarization to astrocyte endfeet requires M23–AQP4. (A) Wide-field and TIRF images of U87-MG cells stably transfected with M1– or M23–AQP4 and stained with antibody to AQP4 and for F-actin with phalloidin. Top panels shows untreated control cells; in bottom panels live cells were subject to detergent extraction by treatment with 0.5% Triton X-100 for 5 min before fixation. (B) Mean cellular AQP4 immunofluorescence staining intensity measured from TIRF images of M1- or M23-transfected cells under control conditions or with 0.5% Triton X-100 treatment before fixation. (n = 5 cells each, ±SE; n.s., P > 0.05; ***, P < 0.001 by t test). (C) TIRF images and ratio image of live U87-MG cells transfected with M1–AQP4–GFP and M23–AQP4–mCherry before and after incubation with 0.5% Triton X-100. (D) Quantification of M1/M23 ratio in transfected U87-MG cells before and after incubation with 0.5% Triton X-100; *, P < 0.05 by paired t test. (E) AQP4 distribution in astrocytes after intracerebral injection of M1– (top) or M23–AQP4 (bottom) adenoviruses into AQP4−/− mice. Left panels show AQP4 distribution in GFAP-labeled astrocytes adjacent to cerebral blood vessels (dotted line), right panels show an enlarged view of AQP4 and GFAP staining in astrocyte end-feet (labeled “E”). (F) Quantification of the ratio of AQP4 staining density in GFAP-labeled processes adjacent to blood vessels versus nonadjacent processes for cells transfected with M1– or M23–AQP4; **, P < 0.01 by t test.

Mentions: AQP4 is highly enriched at astrocyte end-feet, where it is anchored to the basement membrane by interactions with adhesion complexes (Neely et al., 2001). We postulated that M1– and M23–AQP4 may play different roles in this process, reasoning that clustered AQP4 may form more stable interactions with adhesion complexes than individual AQP4 tetramers. Live-cell detergent extraction can be used to assess localization of specific molecules to adhesion sites (Boiko et al., 2007; Zhang et al., 2012), and we applied this method in U87-MG cells expressing M1– or M23–AQP4 and plated on fibronectin-coated coverglasses. Remarkably, more M23–AQP4 remained bound to the substrate after detergent treatment in M23–AQP4- than in M1–AQP4-transfected cells (Fig. 7, A and B), despite complete cell disruption as confirmed by loss of F-actin staining. We also measured the M1/M23 ratio in adherent AQP4 complexes in cells cotransfected with M1–AQP4–GFP and M23–AQP4–mCherry. Detergent-resistant arrays contained both M1– and M23–AQP4, but were enriched in M23–AQP4 (Fig. 7, C and D), supporting the conclusion that large, M23–AQP4-enriched arrays preferentially bind to the extracellular substrate.


Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes.

Smith AJ, Jin BJ, Ratelade J, Verkman AS - J. Cell Biol. (2014)

ECM adhesion and polarization to astrocyte endfeet requires M23–AQP4. (A) Wide-field and TIRF images of U87-MG cells stably transfected with M1– or M23–AQP4 and stained with antibody to AQP4 and for F-actin with phalloidin. Top panels shows untreated control cells; in bottom panels live cells were subject to detergent extraction by treatment with 0.5% Triton X-100 for 5 min before fixation. (B) Mean cellular AQP4 immunofluorescence staining intensity measured from TIRF images of M1- or M23-transfected cells under control conditions or with 0.5% Triton X-100 treatment before fixation. (n = 5 cells each, ±SE; n.s., P > 0.05; ***, P < 0.001 by t test). (C) TIRF images and ratio image of live U87-MG cells transfected with M1–AQP4–GFP and M23–AQP4–mCherry before and after incubation with 0.5% Triton X-100. (D) Quantification of M1/M23 ratio in transfected U87-MG cells before and after incubation with 0.5% Triton X-100; *, P < 0.05 by paired t test. (E) AQP4 distribution in astrocytes after intracerebral injection of M1– (top) or M23–AQP4 (bottom) adenoviruses into AQP4−/− mice. Left panels show AQP4 distribution in GFAP-labeled astrocytes adjacent to cerebral blood vessels (dotted line), right panels show an enlarged view of AQP4 and GFAP staining in astrocyte end-feet (labeled “E”). (F) Quantification of the ratio of AQP4 staining density in GFAP-labeled processes adjacent to blood vessels versus nonadjacent processes for cells transfected with M1– or M23–AQP4; **, P < 0.01 by t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3926963&req=5

fig7: ECM adhesion and polarization to astrocyte endfeet requires M23–AQP4. (A) Wide-field and TIRF images of U87-MG cells stably transfected with M1– or M23–AQP4 and stained with antibody to AQP4 and for F-actin with phalloidin. Top panels shows untreated control cells; in bottom panels live cells were subject to detergent extraction by treatment with 0.5% Triton X-100 for 5 min before fixation. (B) Mean cellular AQP4 immunofluorescence staining intensity measured from TIRF images of M1- or M23-transfected cells under control conditions or with 0.5% Triton X-100 treatment before fixation. (n = 5 cells each, ±SE; n.s., P > 0.05; ***, P < 0.001 by t test). (C) TIRF images and ratio image of live U87-MG cells transfected with M1–AQP4–GFP and M23–AQP4–mCherry before and after incubation with 0.5% Triton X-100. (D) Quantification of M1/M23 ratio in transfected U87-MG cells before and after incubation with 0.5% Triton X-100; *, P < 0.05 by paired t test. (E) AQP4 distribution in astrocytes after intracerebral injection of M1– (top) or M23–AQP4 (bottom) adenoviruses into AQP4−/− mice. Left panels show AQP4 distribution in GFAP-labeled astrocytes adjacent to cerebral blood vessels (dotted line), right panels show an enlarged view of AQP4 and GFAP staining in astrocyte end-feet (labeled “E”). (F) Quantification of the ratio of AQP4 staining density in GFAP-labeled processes adjacent to blood vessels versus nonadjacent processes for cells transfected with M1– or M23–AQP4; **, P < 0.01 by t test.
Mentions: AQP4 is highly enriched at astrocyte end-feet, where it is anchored to the basement membrane by interactions with adhesion complexes (Neely et al., 2001). We postulated that M1– and M23–AQP4 may play different roles in this process, reasoning that clustered AQP4 may form more stable interactions with adhesion complexes than individual AQP4 tetramers. Live-cell detergent extraction can be used to assess localization of specific molecules to adhesion sites (Boiko et al., 2007; Zhang et al., 2012), and we applied this method in U87-MG cells expressing M1– or M23–AQP4 and plated on fibronectin-coated coverglasses. Remarkably, more M23–AQP4 remained bound to the substrate after detergent treatment in M23–AQP4- than in M1–AQP4-transfected cells (Fig. 7, A and B), despite complete cell disruption as confirmed by loss of F-actin staining. We also measured the M1/M23 ratio in adherent AQP4 complexes in cells cotransfected with M1–AQP4–GFP and M23–AQP4–mCherry. Detergent-resistant arrays contained both M1– and M23–AQP4, but were enriched in M23–AQP4 (Fig. 7, C and D), supporting the conclusion that large, M23–AQP4-enriched arrays preferentially bind to the extracellular substrate.

Bottom Line: The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays.Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix.Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, CA 94143.

ABSTRACT
The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1-AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23-AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

Show MeSH
Related in: MedlinePlus