Petrova et al Dynamic changes in AQP5 membrane localization · 29/06/2020 · Petrova et al...

Petrova et al Dynamic changes in AQP5 membrane localization

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Changes to zonular tension alters the subcellular distribution of AQP5 in regions of 2

influx and efflux of water in the rat lens. 3

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Rosica S Petrova*1, Nandini Bavana1, Rusin Zhao1, Kevin L Schey2, Paul J Donaldson1 5

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1Department of Physiology, School of Medical Sciences, New Zealand National Eye Centre, 7

University of Auckland, Auckland, New Zealand; 2Mass Spectrometry Research Center, 8

Vanderbilt University, Nashville, TN, USA 9

Running title: Dynamic changes in AQP5 membrane localization 10

*Corresponding Author: Dr Rosica Petrova 11

Email: [email protected] 12

Word count: 6187 13

Funding: This work was funded by grants from the Royal Society of New Zealand Marsden 14

Fund (16-UOA-251) and the National Eye Institute USA (EY013462-16) 15

Commercial relationships:. Rosica Petrova (none), Nandini Bavana (none), Rusin Zhao 16

(none), Kevin Schey (none), Paul Donaldson (none). 17

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted June 30, 2020. ; https://doi.org/10.1101/2020.06.29.178756doi: bioRxiv preprint

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ABSTRACT 19

Purpose: The lens utilizes circulating fluxes of ions and water that enter the lens at both poles 20

and exit at the equator to maintain its optical properties. We have mapped the subcellular 21

distribution of the lens aquaporins (AQP0, 1, & 5) in these water influx and efflux zones and 22

investigated how their membrane location is affected by changes in tension applied to the lens 23

by the zonules. 24

Methods: Immunohistochemistry using AQP antibodies was performed on axial sections 25

obtained from rat lenses that had been removed from the eye and then fixed, or were fixed in 26

situ to maintain zonular tension. Zonular tension was pharmacologically modulated by 27

applying either tropicamide (increased), or pilocarpine (decreased). AQP labelling was 28

visualized using confocal microscopy. 29

Results: Modulation of zonular tension had no effect on AQP1 or AQP0 labelling in either the 30

water efflux, or influx zones. In contrast, AQP5 labelling changed from membranous to 31

cytoplasmic in response to both mechanical and pharmacologically induced reductions in 32

zonular tension in both the efflux zone, and anterior (but not posterior) influx zone associated 33

with the lens sutures. 34

Conclusions: Altering zonular tension dynamically regulates the membrane trafficking of 35

AQP5 in the efflux and anterior influx zones to potentially change the magnitude of circulating 36

water fluxes in the lens. 37

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KEYWORDS: Lens, water transport, immunohistochemistry, AQP0, AQP1, AQP5, zonular 39

tension. 40

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INTRODUCTION 42

The transparency and refractive properties of the lens are established and maintained by its 43

unique cellular structure and function 1. Structurally, the lens is attached to the ciliary body via 44

the lens zonules, and is bathed on its anterior and posterior surfaces by the aqueous and vitreous 45

humors, from which it exchanges nutrients and waste products. Underneath the capsule the 46

anterior surface of the lens is covered by a single layer of epithelial cells that, at the equatorial 47

margins, divide and differentiate in the lens fiber cells which make up the bulk of the lens 48

(Figure 1A). These fiber cells undergo a process of differentiation that involves not only a 49

change of the molecular profile of the newly differentiated fiber cells, but also a massive 50

elongation of their lateral membrane domains towards the poles of the lens. This process 51

continues until either the apical or basal membrane tips of the fibers meet with the membrane 52

tips of fibers cells from adjacent hemisphere to form the anterior and posterior sutures, 53

respectively2. As fiber cells differentiate, they lose their light scattering organelles and 54

internalize older fiber cells into the lens core or nucleus. Since this process occurs throughout 55

life a gradient of fiber cell age is established with oldest mature fiber cells in the lens nucleus 56

having been laid down during embryogenesis. Since the lens lacks a blood supply, it instead 57

operates an internal microcirculation system (Figure 1A), that acts to maintain the overall tissue 58

architecture of the lens by delivering nutrients, removing waste products, and controlling the 59

volume of lens fiber cells3. This internal microcirculation is generated by circulating ionic and 60

fluid fluxes that enter the lens at both poles via an extracellular pathway, associated with the 61

lens sutures, which directs ions and water towards the deeper regions of the lens (Figure 1A). 62

Ions and water then cross fiber cell membranes before returning to the lens surface via an 63

intercellular outflow pathway mediated by gap junctions. The gap junctions direct the outflow 64

of ions and water to the lens equator where the Na+ pumps, which generate the circulating flux 65


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of Na+ ions that drive the microcirculation system, and other ion and water channels are 66

localized allowing ions and water to cross the membranes of surface cells and leave the lens. 67

68

The intercellular outflow of water through the gap junction generates a substantial hydrostatic 69

pressure gradient4, that is not only remarkably similar amongst different species of lens5, but it 70

is also highly regulated by a dual feedback pathway6. This pathway utilizes the 71

mechanosensitive non-selective ion channels TRPV1 and TRPV4 to sense changes in lens 72

pressure and to activate signaling pathways that reciprocally modulate the activity of Na+ 73

pumps at the lens equator to ensure the pressure gradient is maintained constant. More recently, 74

Chen et al., have shown that altering the zonular tension applied to the lens can alter this 75

constant hydrostatic pressure set point7. Thus, while it is apparent that alterations in 76

ionic/osmotic gradients, via modulation of Na+ pump activity, changes the flow of water 77

(pressure) through the lens, whether the permeability of lens cells to water is also modulated 78

in parallel to changes in the osmotic gradient has yet to be determined. 79

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As in other tissues, the water permeability (PH2O) of lens cell membranes is determined by the 81

profile of the different aquaporin (AQP) proteins that are expressed in the different regions of 82

the lens 8. Three different water channels, AQP0, AQP1 and AQP5 (Figure 1B), all with very 83

different PH2O and regulatory properties are differentially expressed in the lens9-11. AQP1, a 84

constitutively active water channel with a high PH2O, is found only in the epithelium11, 12. AQP0, 85

the most abundant membrane protein in the lens, is found only in the fiber cells13. AQP0 is 86

actually a relative poor water channel9, 14, 15, but has been shown to have additional roles as an 87

adhesion protein16-18 and a scaffolding protein19, 20, and undergoes extensive post-translational 88

cleavage of its C-terminal tail in mature fiber cells15, 21, 22. AQP5, in other tissues, has been 89

shown to act as a regulated water channel with a relatively high PH2O, which when inserted into 90


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the membrane increases the PH2O of epithelial cells23-26. In the lens, immunohistochemical 91

mapping of the subcellular distribution of AQP5 has shown it to be expressed in both the 92

epithelium and throughout the fiber cell mass22, 27. In peripheral fiber cells it is initially found 93

as a cytoplasmic pool of protein that undergoes a transition to the membrane as fiber cells 94

differentiate and become internalized. However, unlike AQP0, AQP5 does not undergo 95

extensive cleavage of its C-terminus in the lens nucleus22. 96

In this study, we have mapped the subcellular distribution of the three lens AQPs to determine 97

whether changes in PH2O contribute to the observed modulation of water transport. We have 98

focused on their relative distributions at the equator and both poles, the regions of the lens 99

involved in water efflux and influx, respectively. Furthermore, in an effort to induce changes 100

in PH2O we have modified zonular tension, which alters hydrostatic pressure7, and have used 101

immunohistochemistry to visualize induced changes in the subcellular location of the AQPs in 102

the water efflux and influx zones. Our results suggest that dynamic changes in the subcellular 103

location of AQP5 may differentially alter the PH2O of fiber cell membranes in the efflux and 104

influx zone and therefore may contribute to the regulation of water transport in the lens. 105

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METHODS 107

Reagents 108

A polyclonal affinity purified anti-AQP0 antibody, directed against the last 17 amino acids of 109

the C-terminus of the human protein, was obtained from Alpha Diagnostic International, Inc. 110

(San Antonio, TX, USA, catalogue number AQP01-A). An affinity purified polyclonal anti-111

AQP5 antibody, directed against the last 17 amino acids of the C-terminus of the rat protein, 112

was obtained from Merck Millipore (Darmstadt, Germany, catalogue number AB15858). A 113

rabbit polyclonal antibody directed against the last 19 amino acids of the C-terminus of the rat 114

AQP1 protein was purchased from Alpha Diagnostic International (San Antonio, Texas, USA). 115

Secondary antibodies (goat anti-Rabbit Alexa 488), and wheat germ agglutinin (WGA) 116

conjugated to a fluorophore (WGA-Alexa 594) for labelling of the cell membrane, were 117

obtained from Thermo Fisher Scientific (Waltham, MA). For labelling of the epithelial and 118

fiber cell nuclei, DAPI was obtained from Sigma–Aldrich (St Louis, MO, USA). Phosphate 119

buffered saline (PBS) was prepared fresh from PBS tablets. Lenses were organ cultured in 120

Artificial Aqueous Humour (AAH) that consisted of (in mM): 125 NaCl, 0.5 MgCl2, 4.5 KCL, 121

10 NaHCO3, 2 CaCl2, 5 Glucose, 10 sucrose, 10 HEPES, pH 7.4, 300mOsml/L. Tropicamide 122

and pilocarpine were used at 1:5 dilution prepared in AAH buffer from 1% w/v eye drops. 123

Unless otherwise stated all other chemicals were from Sigma-Aldrich 124

Lens organ culture 125

All animal experiments were carried out in accordance with the ARVO Statement for the Use 126

of Animals in Ophthalmic and Vision Research and were approved by the University of 127

Auckland Animal Ethics Committee (# 001893). Eyes were enucleated from 21-day-old Wistar 128

rats and subjected to three different preparation protocols that yielded lenses that were either 129

separated from, or attached to, the ciliary muscle by the zonules. In the first preparation, lenses 130


https://www.sciencedirect.com/topics/immunology-and-microbiology/carboxy-terminal-sequence

https://www.sciencedirect.com/topics/immunology-and-microbiology/polyclonal-antibodies

https://www.sciencedirect.com/topics/immunology-and-microbiology/wheat-germ-agglutinin

https://www.sciencedirect.com/topics/immunology-and-microbiology/lens-fiber

https://www.sciencedirect.com/topics/immunology-and-microbiology/lens-fiber

https://www.sciencedirect.com/topics/immunology-and-microbiology/cell-nucleus

https://www.sciencedirect.com/topics/medicine-and-dentistry/dapi

https://www.sciencedirect.com/topics/medicine-and-dentistry/phosphate-buffered-saline

https://www.sciencedirect.com/topics/medicine-and-dentistry/phosphate-buffered-saline

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were separated from the ciliary body by completely removing the lens from the eye. To achieve 131

this, four radiating incisions from the optic nerve head towards the limbus were first made to 132

expose the lens and ciliary body. To release the lens, the zonules were cut with a pair of surgical 133

scissors, the lens was removed from the eye using a glass loop, and the excised lens was organ 134

cultured in AAH for up to 120 minutes before immersing in 0.75% PFA prepared in PBS pH 135

7.4 and fixed overnight at room temperature. In the second preparation, lenses were prepared 136

with the zonules attached by first cutting a small (~2 mm) hole into the cornea of the enucleated 137

eye, to provide a pathway to perfuse the lens with AAH in the absence or presence of either 138

tropicamide or pilocarpine. Lenses were maintained in organ culture for up 120 minutes at 139

37oC in a CO2 incubator (Heracell 150i, Thermo Scientific, USA). Tropicamide causes a 140

relaxation of the ciliary muscle and increases the tension applied to the lens via the zonules, 141

while pilocarpine by inducing a contraction of the ciliary muscle reduces the tension applied 142

to the lens via the zonules. After organ culture, the preparation was perfused with 0.75% PFA, 143

and maintained at room temperature overnight to fix the lens in situ. After fixation lens were 144

dissected free from the surrounding tissue and processed for immunohistochemistry. 145

The third preparation was used to confirm the ability of tropicamide and pilocarpine to alter 146

zonular tension. The drugs were diluted in AAH and applied to the corneal surface of 147

enucleated eyes, and the native three-dimensional structure of the ciliary body, zonules and 148

lens, was preserved by fixing the eyes in 4% paraformaldehyde, while maintaining the 149

intraocular pressure at 18 mmHg28 using the method described by Bassnett29. After fixation, 150

the posterior sclera and retina were removed, and the ciliary body and lens photographed using 151

a stereo microscope (Leica Microsystems, Germany). The distance between the two structures 152

was measured using image analysis software (Adobe Photoshop CC). For each lens ~25 153

measurements were taken to calculate the mean circumferential space distance from at least 154


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three different lenses incubated in the absence and presence of either tropicamide or 155

pilocarpine. 156

Immunohistochemistry 157

Fixed lenses were washed 3 times in PBS pH 7.4 for 10 minutes, and cryoprotected using 3 158

consecutive incubations in 10% and 20% sucrose for 1 hour at room temperature, followed by 159

an overnight incubation in 30% sucrose at 40C30. Cryoprotected lenses were positioned on 160

chucks in an axial orientation, encased in optimal temperature medium (OCT, Japan), and snap 161

frozen for 15 seconds in liquid nitrogen. Axial sections were cut using a Leica CM3050S 162

cryostat (Leica Biosystems, Germany), and consecutive sections of ~14 µm thickness were 163

collected from at least three lenses for each experimental condition to ensure consistency of 164

immunolabelling results. Sections were washed 3 times for 5 minutes in PBS pH 7.4 and 165

blocked for 1 hour in blocking solution (3% normal goat serum, 3% BSA dissolved in PBS, 166

pH 7.4). Excess blocking solution first was removed using tissue paper, before incubating 167

sections in different anti-AQP primary antibodies diluted 1:100 in blocking solution overnight 168

at 40C. Sections were washed 3 times for 5 minutes in PBS pH 7.4, to remove unbound 169

antibody, before incubating sections with a goat anti-rabbit Alexa 488 secondary antibody for 170

3 hours at room temperature. After washing the secondary antibody 3 times in PBS pH 7.4 for 171

5 minutes, sections were incubated at room temperature for 1 hour in the membrane marker 172

WGA and the nucleus marker DAPI (0.25µg/ml), diluted 1:100 and 1:1000 in PBS pH 7.4, 173

respectively. After a final wash in PBS pH 7.4, sections were mounted with a cover slip using 174

an anti-fading agent (Vectashield, Vector Laboratories, San Diego, CA, USA), and imaged 175

using an Olympus FV1000 Fluoview confocal scanning microscope (Tokyo, Japan). The 176

resultant images were prepared using Adobe Photoshop CC software. 177

Statistical Analysis 178


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Experimental means are given as ± standard error of the mean (SE). Statistical significance 179

was tested with the Mann-Whitney U-test, using GraphPad Prism (La Jolla, CA). Statistical 180

significance was set at the α = 0.05 level. 181

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RESULTS 183

In previous studies we have mapped the distribution of AQP021 and AQP522 from the periphery 184

to the center of the lens using sections taken through the equator of the rodent lenses, which 185

were removed from the eye by cutting the zonules that attach the lens to the ciliary body. In 186

this study, we have utilized axial sections to map the subcellular distribution of AQPs in the 187

rat lens, as this orientation allows us to visualize, in the same section, the anterior and posterior 188

poles of the lens that mediate water influx, as well as the lens equator where water efflux occurs 189

(Figure 1). Furthermore, to determine the effects of changes in zonular tension on the 190

subcellular distribution of AQPs, axial sections were obtained from lenses that had been 191

subjected to mechanical and pharmacological manipulations to alter the tension applied to the 192

lens via the zonules. The effects of altering zonular tension on the subcellular localization of 193

the three major AQPs 1, 0, and 5 in the water efflux and influx pathways are presented in turn. 194

The subcellular distribution of lens AQPs in the equatorial water efflux zone 195

As epithelial cells at the lens equator initiate the process of differentiation into fiber cells in the 196

water efflux zone, they change their AQP expression profile (Figure 2). As had been shown 197

previously in lenses removed from the eye by cutting the zonules, AQP1 was expressed only 198

in the epithelial cells that cover the anterior surface of the lens12, with AQP1 labelling being 199

strongly localized to the apical and lateral membrane domains of the cells (Figure 2B). 200

However, as the epithelial cells elongated into fiber cells, AQP1 labelling abruptly disappeared 201

and was completely lacking in secondary fiber cells (Figure 2B). In contrast to AQP1, AQP0 202

protein was not detected in epithelial cells, and was initially only observed in the newly-derived 203

elongating fiber cells as a cytoplasmic punctate labelling pattern, with strong membranous 204

labelling only becoming apparent in fiber cells ~20-25 cell layers in from the capsule (Figure 205

2C). AQP5 labelling was initially predominately cytoplasmic in both the epithelial and newly 206


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differentiated fiber cells (Figure 2D), before becoming localized to the membranes of 207

secondary fiber cells ~150 to 200m in from the capsule in an area just past the bow region 208

where cell nuclei disperse (Figure 2E). In summary, we see a change in expression from AQP1 209

to AQP0 as epithelial cells differentiate into fiber cells, while AQP5 is expressed in both cell 210

types. In the efflux zone, both AQP1 and AQP0 show an exclusive membrane localization that 211

suggests they both contribute to PH2O in this zone, while, the localization of AQP5 will the 212

cytoplasm indicates AQP5 is not actively contributing to the PH2O of epithelial cells and 213

peripheral fiber cells in the efflux zone . However, in deeper differentiating fiber cells the insert 214

of AQP5 in the plasma membrane indicates that in these cells it does contribute to PH2O27. 215

To determine whether altering zonular tension changes the subcellular distribution of the lens 216

AQPs, we organ cultured rat lenses either ex vivo (no zonules) or in situ (with zonules) for 217

different periods of time before fixing the lenses for immunohistochemistry. The pattern of 218

AQP1 (Figure 2B) and AQP0 (Figure 2C) labelling observed in lenses organ cultured in lenses 219

with no zonules attached was not altered in lenses with zonules attached (data not shown). 220

However, the subcellular labelling patterns observed for AQP5 were different in lenses 221

maintained in ex vivo or in situ organ culture (Figure 3). Lenses, maintained in ex vivo organ 222

culture after having their zonules cut, showed an initial predominantly cytoplasmic subcellular 223

localization of AQP5 in both epithelial and fiber cells for up to the first 45 minutes in culture. 224

Then, over the next 60 to 120 minutes the labelling became increasingly associated with the 225

plasma membrane of peripheral fiber cells in the water efflux zone (Figure 3B). In contrast, 226

lenses that were fixed in situ, with their zonules intact, exhibited predominately membranous 227

AQP5 labelling in fiber cells throughout the efflux zone that did not change during the entire 228

120 minutes of organ culture (Figure 3C). Taken together these results suggest that AQP5 in 229

the water efflux zone is normally membranous and that reducing zonular tension results in the 230

rapid removal of AQP5 from the membrane. 231


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To further test this notion, we incubated enucleated eyes in the absence or presence of either 232

tropicamide or pilocarpine, to pharmacologically manipulate iris and ciliary muscle 233

contractility, in order to alter the tension applied to the lens in situ via the zonules (Figure 4). 234

As expected, tropicamide (Figure 4B) and pilocarpine (Figure 4C) evoked dilation and 235

constriction, respectively, of the pupil of the enucleated rat eye, confirming the functionality 236

of drugs on the muscles of the iris. To assess the functionality of the drugs on the ciliary muscle, 237

we first fixed the entire globe, before removing the posterior tissues of the eye to reveal the 238

ciliary body and lens in order to allow the distance between the ciliary process and lens, the 239

circumlental space, to be measured. Under control conditions the circumlental space was 240

151.51 + 2.3 µm (n = 5), which was significantly increased in the presence of tropicamide by 241

11% to 167.67 + 1.4 µm (n = 3), and significantly decreased by 14% to 129.03 + 2.5 µm after 242

incubation in pilocarpine (n = 3) (Figure 4H). Having demonstrated that we could 243

pharmacologically modulate ciliary muscle contractility to alter the distance between the ciliary 244

processes and the lens, we used these pharmacological tools to alter the tension applied to the 245

lens via the zonules. 246

If the assumption that ciliary muscle contractility alters zonular tension in our experimental 247

model is true, we would expect that narrowing of the circumlental space induced by pilocarpine 248

should have a similar effect on the subcellular distribution of AQP5 as that seen in lenses fixed 249

immediately after having their zonules mechanically cut to release the tension applied to the 250

lens. To test this notion immunohistochemistry for AQP5 was performed on lenses fixed in situ 251

following incubation in the absence or presence of either tropicamide, or pilocarpine (Figure 252

5). We found that increasing the tension applied to the lens via the zonules by the application 253

of tropicamide had no effect on the membrane location of AQP5 (Figure 5C), compared to 254

control lenses with intact zonules (Figure 5B). In contrast, eyes incubated in pilocarpine that 255

reduces the tension applied to the lens exhibited AQP5 labelling that was predominately 256


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associated with the cytoplasm of fiber cells in the water efflux zone (Figure 5D). In summary, 257

it appears that both mechanically and pharmacologically reducing the tension applied to the 258

lens switches AQP5 labelling from the membrane to cytoplasm, which suggests AQP5 259

functionality can be dynamically regulated to alter water efflux at the lens equator. In the next 260

sections we investigate whether similar changes in the subcellular distribution of lens AQPs 261

occurred in the water influx zones located at the anterior and posterior poles. 262

263

The subcellular distribution of lens AQPs in the water influx zone 264

Experiments that have utilized Ussing chambers31 and MRI measurements of heavy water 265

penetration into the lens32, have shown that water preferentially enters the lens at its anterior 266

and posterior poles. At the poles, fiber cells from adjacent hemispheres meet to form the lens 267

sutures2, which in the rat lens form a Y-shaped structure. The sutures form an extracellular 268

pathway that links the mature fiber cells in the center of the lens to the aqueous and vitreous 269

humors that bathe the anterior and posterior poles of the lens, respectively. As such the sutures 270

represent a pathway to direct ion and fluid movement towards the internalized mature fiber 271

cells33. In axial sections it is possible to visualize the sutures as a line extending from the surface 272

to the core of the lens, but it is difficult to observe the anterior and posterior sutures in the same 273

section as they are offset from each other by 60o34 . In this study, we have investigated the 274

distribution of the AQPs in anterior (IA) and posterior (IP) influx zones by mapping the 275

subcellular distribution of the three AQPs along the two suture lines that extend from the 276

anterior and posterior poles to the lens core. To facilitate this comparison we have designated 277

three spatial regions in the outer cortex (IA1; IP1), inner cortex (IA2; IP2) and core (IA3; IP3) 278

from which higher resolution images have been taken to enable comparison between the 279


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labelling obtained from lenses exposed to different degrees of zonular tension (Figure 1B). 280

Results from the anterior and posterior poles are presented in turn. 281

AQP distributions along the posterior suture: Since AQP1 is not present in the fiber cells 282

below the germinative zone, we only examined the distribution of AQP0 and AQP5 in the 283

posterior influx zone. We found that in lenses with their zonules cut, AQP0 was localized to 284

the lateral membranes of fiber cells and was concentrated in the basal tips of fibers that interact 285

to form the sutures in the outer cortex of the lens (Figure 6B, IP1). In contrast, AQP5 was 286

cytoplasmic with intermittently sparse clusters of puncta on the lateral membranes of fiber 287

cells, but no localization of AQP5 was observed at the basal tips of fiber cells that interface to 288

form the posterior suture (Figure 6C, IP1). In the inner cortex AQP0 remained membranous 289

and associated with the suture, while AQP5 was predominately associated with lateral 290

membranes, but not the posterior suture (Figure 6B&C, IP2). In mature fiber cells located in 291

the lens core AQP0 labelling was abolished, as expected by the C-terminal cleavage of the 292

AQP0 protein as AQP0 protein undergoes a cleavage of its cytoplasmic C-terminus tail, which 293

removes the epitope detected by the AQP0 antibody used in this study21. In contrast, strong 294

AQP5 labelling was associated with both the lateral membranes and the sutures in the lens 295

nucleus (Figure 6B&C, IP3). Repeating these experiments using lenses fixed in situ to maintain 296

zonular tension had no effect on the subcellular distribution of AQP0 (data not shown) or AQP5 297

(Figure 6D) in all regions of interest in the posterior water influx zone. Similarly, 298

pharmacological modulation of zonular tension using tropicamide or pilocarpine had no effect 299

on the distribution of AQP5 in the posterior influx zone (data not shown). 300

AQP distributions along the anterior suture: As shown previously for the efflux zone, AQP1 301

was only expressed in the epithelial cells that cover the anterior surface of the lens, and hence 302

it was not associated with the anterior suture (Figure 7B, IA1), nor was it expressed in the 303

deeper regions of the lens (Figure 7B, IA2 & IA3). AQP0 labelling around anterior suture was 304


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essentially similar to that seen in the posterior suture. In the outer cortex of lenses with zonules 305

cut (Figure 7C) AQP0 was not present in the epithelial cells, was localized to the lateral 306

membranes of fiber cells and was present in the sutures formed from the apical membrane 307

domains (Figure 7C, IA1). AQP0 labelling remained membranous in the fiber cells and 308

strongly localized to the suture in the inner cortex (Figure 7C, IA2). However, as expected 309

AQP0 labelling was not detected in the lens core due to the loss of the antibody epitope (Figure 310

7C, IA3). AQP5 labelling in the anterior influx zone of lenses with their zonules cut, was 311

essentially similar to that seen in the posterior influx zone. AQP5 labelling was strongly 312

cytoplasmic in the epithelial cells, with a mixed cytoplasmic and membranous localization in 313

the lateral membranes of fiber cells in the outer cortex, and no labelling of the apical 314

membranes associated with the sutures in this region of the lens (Figure 7D, IA1). In the inner 315

cortex AQP5 labelling was associated with the lateral membranes, but was absent from the 316

apical tips at the sutures (Figure 7, IA2), while in the core AQP5 remained membranous in the 317

lateral domains, and was found strongly associated with the sutures (Figure 7D, IA3). However, 318

changes in zonular tension did differentially alter the AQP5 distribution in the anterior influx 319

zone. While no changes in AQP5 labelling were seen in regions IA1 and IA3, of lenses fixed 320

in situ with their zonules attached, an increased association of AQP5 with the sutures was 321

observed in region IA2 (Figure 7E). To confirm that a change in zonular tension is the 322

underlying mechanism regulating this observed change in AQP5 labelling in this region of the 323

anterior influx zone, we treated eyes with tropicamide or pilocarpine (Figure 8). We found that 324

in lenses with their zonules attached tropicamide had no effect on AQP5 labelling in the 325

anterior suture (Figure 8B, IA2). However, reducing zonular tension via the application of 326

pilocarpine abolished AQP5 labelling associated with the suture in the inner cortical region of 327

the anterior influx zone (Figure 8C, IA2). In summary, it appears that AQP5 is differentially 328

associated with the apical and basal membrane domains that form anterior and posterior 329


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sutures, respectively, in the water influx zone, and in the anterior pole this association with the 330

apical membrane domain can be modified by changes in zonular tension. 331

332


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DISCUSSION 333

Cells move water by actively transporting ions and solutes to establish osmotic gradients that 334

drive the passive diffusion of water through water channels formed from the aquaporin family 335

of proteins35, 36. The lens expresses at least three AQPs with very different PH2O and regulation 336

that show spatially distinct patterns of expression and posttranslational modifications. In this 337

study we have utilized immunohistochemistry to visualize the subcellular distribution of the 338

lens AQPs in two spatially discrete areas of the lens that have been shown to be associated 339

water influx and efflux31, 32. Our results have confirmed the restriction of AQP1 labelling to 340

the lens epithelium (Figures 2B, 7B), the ubiquitous labelling of AQP0 in the membranes of 341

lens fiber cells (Figures 2C, 6B, 7B), and the loss of AQP0 labelling in the lens nucleus due to 342

the cleavage of the C-terminus of the AQP0 protein (Figure 6B, 7B). In addition, we have 343

shown that these labelling patterns for both AQP1 and AQP0 are unaffected by either 344

mechanical or pharmacological modulation of zonular tension. We have also confirmed the 345

cytoplasmic labelling for AQP5 in epithelial and peripheral fiber cells (Figure 2D) and 346

membrane labelling in deeper fiber cells (Figure 2E), originally observed in equatorial sections 347

obtained from lenses removed from the eye by cutting the zonules22. However, in addition we 348

have shown that this labelling pattern is altered by fixing lenses in situ to maintain the zonular 349

tension applied to the lens (Figure 3C). In the influx zone we have shown that AQP5 is 350

preferentially localized at the tips of fiber cells that form the anterior (Figure 7D, E), but not 351

the posterior sutures (Figures 6C, D). Furthermore, the membrane localization of AQP5 to the 352

anterior suture was decreased by either the mechanical (Figure 7E, region A2) or 353

pharmacological (Figure 8D, region A2) reductions in zonular tension applied to the lens. 354

These changes in the subcellular location of AQP5 in response to change in zonular tension, 355

are summarized in Figure 9. Taken together our finding suggest AQP5 acts as a regulated water 356

channel that can dynamically regulate the PH2O of lens fiber cells in the anterior influx and 357


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equatorial efflux zones to potentially modulate lens water transport in response to changes in 358

zonular tension. 359

The existence of a pool of cytoplasmic membrane proteins that translocate to the plasma 360

membrane of fiber cells, either as a function of fiber cell differentiation or dynamically in 361

response to applied stimuli, to confer a specific function required by lens fiber cells, appears to 362

be a reoccurring theme in the lens37, 38. In this study, we have shown that in lenses fixed in situ 363

the default subcellular location for AQP5 is the membrane, and that upon reducing the zonular 364

tension AQP5 is removed from the membrane, presumably to reduce the PH2O of fiber cell 365

membranes in the anterior and equatorial zones of water influx and efflux, respectively. 366

Interestingly, this removal of AQP5 from the membrane is only transient in lenses organ 367

cultured without their zonules attached (Figure 3B). In an earlier study, we showed a similar 368

increase in AQP5 membrane labelling in the equatorial efflux zone and, in addition, showed 369

that this increase in membrane labelling was associated with an increase in the Hg+-sensitive 370

PH2O measured in fiber membrane vesicles derived from organ cultured rat lenses27. This earlier 371

study indicates that the changes in the subcellular location of AQP5 labelling observed using 372

immunohistochemistry in the current study are associated with changes in PH2O and support the 373

suggestion that AQP5 functions as a regulated water channel in the anterior influx and 374

equatorial efflux zones of the rat lens. 375

Despite its potential contributions to the regulation of lens water transport, it was surprising to 376

see that the deletion of the AQP5 gene did not induce a cataract in AQP5-KO lenses39. 377

However, it does appear that when AQP5-KO lenses are organ cultured under hyperglycaemic 378

conditions they are more susceptible to the development of cataract, than wild type lenses39. 379

This is most probably due to the inability of the AQP5-KO lens to alter its PH2O in response to 380

the osmotic stress induced by the hyperglycaemia, supporting the suggestion that AQP5 plays 381

a role in regulating water fluxes under both steady state conditions and under conditions of 382


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stress. Consistent with this idea, an increased expression of AQP5 was observed in epithelial 383

cells removed from patients undergoing cataract surgery40. In these patients not only were the 384

expression levels of both AQP5 and AQP1 increased, but AQP5 was found to be more strongly 385

localised to the membranes of lens epithelial cells obtained from cataract patients relative to 386

epithelial cells obtained from age matched non-cataractic lenses. Thus, it appears that as a 387

regulated water channel AQP5 can respond to imposed stresses to modulate water transport in 388

an effort to maintain fluid homeostasis and preserve lens transparency. 389

In other epithelial tissues, AQP5 has been also shown to act as a regulated water channel that 390

inserts into the apical plasma membrane following phosphorylation of the channel through the 391

cAMP-dependent PKA pathway23. In addition, exposure to hypertonic challenge was shown to 392

upregulate AQP5 protein expression through an extracellular signal-regulated kinase (ERK)-393

dependent pathway in mouse lung epithelial (MLE-15) cells25. From the current study, we can 394

add the tension applied to the lens via the zonules as a stimulus capable of regulating the 395

trafficking of AQP5 to the membrane. The observation of this phenomenon raises questions 396

about how changes in zonular tension are sensed and translated to alterations in AQP5 397

membrane trafficking. Again in other tissues, a synergistic association between the mechano-398

sensitive TRPV4 and aquaporin water channels has been shown to effect changes in fluid 399

transport to preserve cell volume41-43. In the lens, TRPV4 and TRPV1 channels have been 400

shown to not only reciprocally regulate the hydrostatic pressure generated by water flow 401

through gap junction channels6, but also to transduce changes to the magnitude of this pressure 402

gradient induced by pharmacologically modulating the zonular tension applied to the lens7. 403

Furthermore, changes in zonular tension induced either, mechanically by cutting the zonules44, 404

or pharmacologically via application of tropicamide or pilocarpine, also altered the subcellular 405

location of TRPV1 and TRPV4 (data presented at the 6th International Conference on the Lens 406

by Nakazawa et al., conference proceedings unpublished). Taken together these observations 407


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suggest that in addition to the modulation of the osmotic gradients that drive the transport of 408

water6, 45, changes to PH2O are also required to maintain the gradient in hydrostatic pressure that 409

has been measured in all lenses studied to date5. Finding a link between TRPV1/4 mediated 410

signalling pathways and the membrane trafficking of AQP5 in lens fiber cells will be a focus 411

of ongoing work. 412

413

While similar effects on the membrane localization of AQP5, and presumably the PH2O, were 414

evident in the anterior influx and equatorial efflux zones following changes in zonular tension, 415

AQP5 labelling in the posterior influx zone was unaffected, suggesting that changes in zonular 416

tension may differentially affect water influx at the anterior and posterior poles. Structurally 417

the differences in AQP5 localization at the anterior and posterior sutures can be explained by 418

the maintenance of the apical-basal membrane polarity of lens epithelial cells as they 419

differentiate and elongate into fiber cells that then become internalized (Figure 9). The anterior 420

suture is formed by the apical domains of fiber cells that originate from opposing sides of the 421

lens, while the basal domains form the posterior suture46. From the current study it would 422

appear that in the outer and inner cortical regions of the lens the apical domains contain AQP5, 423

but the basal domains do not (Figure 6 & 7). Interestingly, in the oldest fiber cells in the lens 424

nucleus this apical-basal polarity is lost and AQP5 is associated with the posterior suture in the 425

nucleus (Figure 7, IP3). This change could be associated with the loss of components of the 426

lens cytoskeleton in the lens nucleus that mediate the anchoring of membrane proteins to 427

specific membrane domains47-51. 428

While it is apparent that the presence of a regulated water channel such as AQP5 can contribute 429

to the maintenance of steady state water transport and water content, the functional significance 430

of the absence of AQP5 in the posterior influx zone is not immediately obvious. However, 431

recent studies performed on zebrafish lenses may provide some insight into the significance of 432


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this differential expression AQP5 in the anterior and posterior influx zones. In the zebrafish 433

AQP0 exists in as two functionally divergent isoforms: AQP0a and AQP0b 52-55. While both 434

AQP0a and AQP0b appear to permeate water, only AQP0b functions as an adhesion molecule 435

56. Furthermore, it has been found that the knockout of AQP0a resulted in an anterior polar 436

opacity, suggesting that the water transport function of AQP0a is essential for the formation 437

and maintenance of the anterior suture, but not the posterior suture55. Furthermore, in the wild 438

type zebrafish it has been shown that a shift in the location of lens nucleus from an initial 439

anterior position in zebrafish larval to the centre in adult fish is necessary for the development 440

of normal lens optics, and that this process was prevented by the deletion of AQP0a55. If we 441

speculate that in the mammalian lens AQP5 and AQP0 are equivalent to zebrafish AQP0a and 442

AQP0b, respectively, then the presence of AQP5 in the anterior suture may be playing a similar 443

role in setting the optical properties of the mammalian lens. 444

One caveat, however, is that unlike the zebrafish lens, which is round, the mammalian lens has 445

different anterior and posterior radii of curvature. Hence, rather than changing the position 446

(centralisation) of the high refractive index nucleus and therefore lens optical power, as was 447

observed in the developing zebrafish lens, we instead envisage changes to AQP5 mediated 448

water influx at the anterior suture would alter the radius of the curvature of the anterior surface 449

to change the power of the mammalian lens. If this is correct, then the mechanically induced 450

changes in lens shape caused by changes in zonular tension would be amplified by the changes 451

in AQP5 mediated water fluxes in the anterior water influx zone to change the optical power 452

of the lens. 453

In the rat, which does not appear to accommodate, we envisage that as the lens grows associated 454

changes to zonular tension would produce changes to the steady state optics of the lens to 455

ensure light will remain correctly focussed on the retina. In the human lens, which does 456

accommodate, studies have shown that the anterior curvature of the lens is altered to a greater 457


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degree than the posterior curvature57. In the accommodated lens, the radius of the anterior lens 458

surface is ~4.7 times greater than the posterior surface, and the anterior surface becomes 459

significantly more hyperbolic58. Whether AQP5 plays a role in the process of accommodation 460

in the human lens will be an interesting area to pursue in future work. 461

In summary, using a series simple immunohistochemical labelling experiments, guided by our 462

knowledge of water transport in the lens, we have shown that a decrease in zonular tension 463

removes AQP5 from the membranes of fibre cells in the equatorial water efflux zone and the 464

anterior water influx zones. Studying how these observations impact the regulation of lens 465

water transport, which is emerging as being essential to the maintenance of the transparent and 466

refractive properties of the lens, has the potential to increase our understand of not only the 467

normal function of the lens, but also how dysfunction of lens water transport can result in lens 468

cataract. 469

470


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1. Bassnett S, Shi Y, Vrensen GFJM. Biological glass: structural determinants 471 of eye lens transparency. Philosophical Transactions of the Royal Society of London 472

B: Biological Sciences 2011;366:1250-1264. 473 2. Kuszak JR, Zoltoski RK, Tiedemann CE. Development of lens sutures. Int J 474

Dev Biol 2004;48:889-902. 475 3. Mathias RT, Kistler J, Donaldson P. The lens circulation. Journal of 476 Membrane Biology 2007;216:1-16. 477

4. Gao J, Sun X, Moore LC, White TW, Brink PR, Mathias RT. Lens intracellular 478 hydrostatic pressure is generated by the circulation of sodium and modulated by 479

gap junction coupling. The Journal of General Physiology 2011;137:507-520. 480 5. Gao J, Sun X, Moore LC, Brink PR, White TW, Mathias RT. The effect of size 481 and species on lens intracellular hydrostatic pressure. Investigative 482

Ophthalmology & Visual Science 2013;54:183. 483 6. Gao J, Sun X, White TW, Delamere NA, Mathias RT. Feedback Regulation of 484

Intracellular Hydrostatic Pressure in Surface Cells of the Lens. Biophysical Journal 485 2015;109:1830-1839. 486 7. Chen Y, Gao J, Li L, et al. The Ciliary Muscle and Zonules of Zinn Modulate 487

Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential 488 Vanilloid Channels. Investigative ophthalmology & visual science 2019;60:4416-489

4424. 490 8. Kozono D, Yasui M, King LS, Agre P. Aquaporin water channels: atomic 491

structure molecular dynamics meet clinical medicine. The Journal of Clinical 492 Investigation 2002;109:1395-1399. 493 9. Varadaraj K, Kushmerick C, Baldo GJ, Bassnett S, Shiels A, Mathias RT. The 494

role of MIP in lens fiber cell membrane transport. The Journal of Membrane Biology 495 1999;170:191-203. 496

10. Grey AC, Walker KL, Petrova RS, et al. Verification and spatial localization 497 of aquaporin-5 in the ocular lens. Experimental Eye Research 2013;108:94-102. 498 11. Ruiz-Ederra J, Verkman AS. Accelerated cataract formation and reduced 499

lens epithelial water permeability in aquaporin-1-deficient mice. Investigative 500 Ophthalmology & Visual Science 2006;47:3960-3967. 501

12. Varadaraj K, Kumari SS, Mathias RT. Functional expression of aquaporins 502 in embryonic, postnatal, and adult mouse lenses. Developmental Dynamics 503 2007;236:1319-1328. 504

13. Bok D, Dockstader J, Horwitz J. Immunocytochemical localization of the lens 505 main intrinsic polypeptide (MIP26) in communicating junctions. The Journal of Cell 506

Biology 1982;92:213-220. 507 14. Yang B, Verkman AS. Water and glycerol permeabilities of aquaporins 1-5 508 and MIP determined quantitatively by expression of epitope-tagged constructs in 509

Xenopus oocytes. Journal of Biological Chemistry 1997;272:16140-16146. 510 15. Ball LE, Little M, Nowak MW, Garland DL, Crouch RK, Schey KL. Water 511

permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the 512 aging human lens. Investigative Ophthalmology & Visual Science 2003;44:4820-513 4828. 514

16. Lo W-K, Harding CV. Square arrays and their role in ridge formation in 515 human lens fibers. Journal of ultrastructure research 1984;86:228-245. 516

17. Zampighi GA, Hall JE, Ehring GR, Simon SA. The structural organization and 517 protein composition of lens fiber junctions. The Journal of Cell Biology 518 1989;108:2255-2275. 519

18. Gonen T, Cheng Y, Sliz P, et al. Lipid-protein interactions in double-layered 520 two-dimensional AQP0 crystals. Nature 2005;438:633-638. 521


https://doi.org/10.1101/2020.06.29.178756


24

19. Rose KML, Gourdie RG, Prescott AR, Quinlan RA, Crouch RK, Schey KL. The 522 C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and 523

CP49. Investigative Ophthalmology & Visual Science 2006;47:1562-1570. 524 20. Wang Z, Schey KL. Aquaporin-0 interacts with the FERM domain of 525

ezrin/radixin/moesin proteins in the ocular lens. Investigative Ophthalmology & 526 Visual Science 2011;52:5079. 527 21. Grey AC, Li L, Jacobs MD, Schey KL, Donaldson PJ. Differentiation-528

dependent modification and subcellular distribution of aquaporin-0 suggests 529 multiple functional roles in the rat lens. Differentiation 2009;77:70-83. 530

22. Petrova RS, Schey KL, Donaldson PJ, Grey AC. Spatial distributions of AQP5 531 and AQP0 in embryonic and postnatal mouse lens development. Experimental Eye 532 Research 2015;132:124-135. 533

23. Woo J, Chae YK, Jang SJ, et al. Membrane trafficking of AQP5 and cAMP 534 dependent phosphorylation in bronchial epithelium. Biochemical and biophysical 535

research communications 2008;366:321-327. 536 24. Ishikawa Y, Eguchi T, Skowronski MT, Ishida H. Acetylcholine acts on M 3 537 muscarinic receptors and induces the translocation of aquaporin5 water channel 538

via cytosolic Ca 2+ elevation in rat parotid glands. Biochemical and Biophysical 539 Research Communications 1998;245:835-840. 540

25. Hoffert JD, Leitch V, Agre P, King LS. Hypertonic induction of aquaporin-5 541 expression through an ERK-dependent pathway. Journal of Biological Chemistry 542

2000;275:9070-9077. 543 26. Moore M, Ma T, Yang B, Verkman A. Tear secretion by lacrimal glands in 544 transgenic mice lacking water channels AQP1, AQP3, AQP4 and AQP5. 545

Experimental eye research 2000;70:557-562. 546 27. Petrova RS, Webb KF, Vaghefi E, Walker K, Schey KL, Donaldson PJ. 547

Dynamic functional contribution of the water channel AQP5 to the water 548 permeability of peripheral lens fiber cells. American Journal of Physiology-Cell 549 Physiology 2017;314:ajpcell. 00214.02017. 550

28. Wang W-H, Millar JC, Pang I-H, Wax MB, Clark AF. Noninvasive 551 measurement of rodent intraocular pressure with a rebound tonometer. 552

Investigative ophthalmology & visual science 2005;46:4617-4621. 553 29. Bassnett S. A method for preserving and visualizing the three-dimensional 554 structure of the mouse zonule. Experimental eye research 2019;185:107685. 555

30. Jacobs MD, Donaldson PJ, Cannell MB, Soeller C. Resolving morphology and 556 antibody labeling over large distances in tissue sections. Microscopy Research and 557

Technique 2003;62:83-91. 558 31. Candia OA, Mathias R, Gerometta R. Fluid circulation determined in the 559 isolated bovine lens. Investigative Ophthalmology & Visual Science 2012;53:7087. 560

32. Vaghefi E, Pontre BP, Jacobs MD, Donaldson PJ. Visualizing ocular lens fluid 561 dynamics using MRI: manipulation of steady state water content and water fluxes. 562

American Journal of Physiology-Regulatory, Integrative and Comparative 563 Physiology 2011;301:R335-R342. 564 33. Zampighi GA, Simon SA, Hall JE. The specialized junctions of the lens. 565

Internaional Review of Cytology 1992;136:185-225. 566

34. Al‐Ghoul KJ, Lindquist TP, Kirk SS, Donohue ST. A Novel Terminal Web‐Like 567 Structure in Cortical Lens Fibers: Architecture and Functional Assessment. The 568 Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology 569

2010;293:1805-1815. 570 35. Verkman AS, Ruiz-Ederra J, Levin MH. Functions of aquaporins in the eye. 571 Progress in Retinal and Eye Research 2008;27:420-433. 572


https://doi.org/10.1101/2020.06.29.178756


25

36. Schey KL, Wang Z, Wenke JL, Qi Y. Aquaporins in the eye: expression, 573 function, and roles in ocular disease. Biochimica et Biophysica Acta (BBA)-General 574

Subjects 2014;1840:1513-1523. 575 37. Donaldson PJ, Grey AC, Merriman-Smith BR, et al. Functional imaging: new 576

views on lens structure and function. Clinical and Experimental Pharmacology and 577 Physiology 2004;31:890-895. 578 38. Donaldson PJ, Lim J. Membrane transporters. Ocular Transporters in 579

Ophthalmic Diseases and Drug Delivery: Springer; 2008:89-110. 580 39. Kumari SS, Varadaraj K. Aquaporin 5 knockout mouse lens develops 581

hyperglycemic cataract. Biochemical and biophysical research communications 582 2013;441:333-338. 583 40. Barandika O, Ezquerra-Inchausti M, Anasagasti A, et al. Increased 584

aquaporin 1 and 5 membrane expression in the lens epithelium of cataract 585 patients. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 586

2016;1862:2015-2021. 587 41. Liu X, Bandyopadhyay B, Nakamoto T, et al. A Role for AQP5 in Activation 588 of TRPV4 by Hypotonicity CONCERTED INVOLVEMENT OF AQP5 AND TRPV4 IN 589

REGULATION OF CELL VOLUME RECOVERY. Journal of Biological Chemistry 590 2006;281:15485-15495. 591

42. Benfenati V, Caprini M, Dovizio M, et al. An aquaporin-4/transient receptor 592 potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in 593

astrocytes. Proceedings of the National Academy of Sciences 2011;108:2563-594 2568. 595 43. Taguchi D, Takeda T, Kakigi A, Takumida M, Nishioka R, Kitano H. 596

Expressions of aquaporin‐2, vasopressin type 2 receptor, transient receptor 597 potential channel vanilloid (TRPV) 1, and TRPV4 in the human endolymphatic sac. 598

The Laryngoscope 2007;117:695-698. 599 44. Nakazawa Y, Donaldson PJ, Petrova RS. Verification and spatial mapping of 600 TRPV1 and TRPV4 expression in the embryonic and adult mouse lens. 601

Experimental eye research 2019;186:107707. 602 45. Shahidullah M, Mandal A, Delamere NA. Activation of TRPV1 channels leads 603

to stimulation of NKCC1 cotransport in the lens. American Journal of Physiology-604 Cell Physiology 2018;315:C793-C802. 605 46. Zampighi GA, Eskandari S, Kreman M. Epithelial organization of the 606

mammalian lens. Experimental Eye Research 2000;71:415-435. 607 47. Sandilands A, Prescott AR, Carter J, et al. Vimentin and CP49/filensin form 608

distinct networks in the lens which are independently modulated during lens fibre 609 cell differentiation. Journal of cell science 1995;108:1397-1406. 610

48. Blankenship TN, Hess JF, FitzGerald PG. Development-and differentiation-611 dependent reorganization of intermediate filaments in fiber cells. Investigative 612 ophthalmology & visual science 2001;42:735-742. 613

49. FitzGerald PG. Lens intermediate filaments. Experimental eye research 614 2009;88:165-172. 615

50. Oka M, Kudo H, Sugama N, Asami Y, Takehana M. The function of filensin 616 and phakinin in lens transparency. Molecular vision 2008;14:815. 617 51. Xu L, Overbeek PA, Reneker LW. Systematic analysis of E-, N-and P-618

cadherin expression in mouse eye development. Experimental eye research 619 2002;74:753-760. 620

52. Froger A, Németh-Cahalan K, Kalman K, Schilling TF, Hall JE. Knockdown 621 of Zeb1-AQP0 or Zeb2-AQP0 Leads to Cataract Formation in Zebrafish. 622 Investigative Ophthalmology & Visual Science 2008;49:3170-3170. 623


https://doi.org/10.1101/2020.06.29.178756


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53. Froger A, Clemens D, Kalman K, Németh-Cahalan KL, Schilling TF, Hall JE. 624 Two distinct aquaporin 0s required for development and transparency of the 625

zebrafish lens. Investigative Ophthalmology & Visual Science 2010;51:6582. 626 54. Clemens DM, Németh-Cahalan KL, Trinh LT, Zhang T, Schilling TF, Hall JE. 627

In Vivo Analysis of Aquaporin 0 Function in Zebrafish: Permeability Regulation Is 628 Required for Lens TransparencyIn Vivo Analysis of Aquaporin. Investigative 629 Ophthalmology & Visual Science 2013;54:5136-5143. 630

55. Vorontsova I, Gehring I, Hall JE, Schilling TF. Aqp0a regulates suture 631 stability in the zebrafish lens. Investigative ophthalmology & visual science 632

2018;59:2869-2879. 633 56. Vorontsova I, Vallmitjana A, Nakazawa Y, et al. Aquaporin 0A is Required 634 for Water Homeostasis in the Zebrafish Lens In Vivo. Biophysical journal 635

2020;118:167a. 636 57. Dubbelman M, Van der Heijde GL. The shape of the aging human lens: 637

curvature, equivalent refractive index and the lens paradox. Vision research 638 2001;41:1867-1877. 639 58. Dubbelman M, Van der Heijde GL, Weeber HA. Change in shape of the aging 640

human crystalline lens with accommodation. Vision research 2005;45:117-132. 641

642

643


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FIGURE LEGENDS 644

Figure 1. Schematic diagrams illustrating lens structure and function and the distribution 645

of AQPs in different regions of the lens. (A) 3D diagram of the lens showing ion and water 646

fluxes coming into the lens core (yellow) via an extracellular route located at the anterior and 647

posterior sutures (blue arrows). Ions and water cross fiber cell membranes before travelling via 648

an intercellular pathway mediated by gap junction channels (dark blue arrow) to exit the lens 649

at the equator. (B) Diagram of an axial section of the lens showing subcellular distributions of 650

the lens water channels (AQP) in the different regions of the lens. AQP1 (red) is restricted to 651

the membranes of the lens epithelium. AQP0 (left) is found in the membranes of lens fiber cells 652

across all areas of the lens, but in the core of the lens the C-terminal tail is cleaved. AQP5 653

(right) is also found throughout all regions of the lens, but in the epithelial (not shown) and 654

peripheral differentiating fiber (purple) cells it is associated with the cytoplasm. In deeper 655

regions of the outer cortex AQP5 becomes associated with the plasma membrane (blue), and 656

this labelling extends into the lens core. In this study we focused on the subcellular distributions 657

of the three lens AQPs at the equator, which is associated with water efflux (E1), and the 658

anterior (IA1, IA2, IA3) and posterior (IP1, IP2, IP3) poles which mediate water influx. 659

Adapted with permission from Shi et al. (2009). 660

661

662

663

664

665

666


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Figure 2. Subcellular localization of lens AQPs in the efflux zone of rat lenses in the 667

absence of zonular tension. (A) Image montage of the water efflux zone taken from an axial 668

section of a rat lenses that had been removed from the eye by first cutting the zonules, and was 669

then placed immediately into fixative. The section was labelled with the membrane marker 670

WGA (red) and the nuclei marker DAPI (blue). Boxes indicate that areas from which the higher 671

resolution images shown in images B-E were taken from to investigate the subcellular 672

distribution for each lens AQP (green). (B) AQP1 labelling is localized to the membranes of 673

the epithelial cells but disappears as the epithelial cells differentiate into fiber cells. (C) AQP0 674

labelling was absent from epithelial cells, but became apparently initially as diffuse punctate 675

labelling as epithelial cell differentiated into fiber cells, and became strongly associated with 676

the membranes of fiber cells are later stages of differentiation deeper localized secondary fiber 677

cells. (D) AQP5 labeling was initially strongly cytoplasmic in the epithelial cells and newly 678

differentiated fiber cells and only became membranous in differentiating fiber cells at ~150 µm 679

distance from the capsule of the lens (E). 680

681

682

683

684

685

686

687

688

689


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Figure 3. Effects of mechanically altering zonular tension on the subcellular localization 690

of AQP5 in the efflux zone of the rat lens. (A) Image montage of the water efflux zone taken 691

from a representative axial section of a rat lenses labelled with the membrane marker WGA 692

(red) and the nuclei marker DAPI (blue). The box indicates the area from which high resolution 693

images (B & C) were captured to monitor the time course of changes to the subcellular 694

distribution of AQP5 (green) over a period of up to 120 minutes, in lenses that had been 695

removed from the eye by cutting the zonules (B), or in lenses maintained in situ with their 696

zonules intact (C). Top panels in B & C show nuclei, membrane and AQP5 labelling, while 697

bottom panels show only nuclei and AQP5 labelling. In lenses maintained in organ culture with 698

their zonules cut (B) the subcellular localization of AQP5 changes from a cytoplasmic to a 699

membranous labelling pattern over time. While in lenses in which the zonular tension is 700

maintained (C), AQP5 labelling is associated with the membrane and this labelling does not 701

change over time in organ culture. 702

703

704

705

706

707

708

709

710

711


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Figure 4. Pharmacological modulation of zonular tension of the rat lens. (A-C) Images 712

looking down on the anterior surface of enucleated rat eyes showing the pupil diameter in 713

untreated eyes (A), and the increase and decrease following treatment with either tropicamide 714

(B) or pilocarpine (C), respectively, for 60 minutes. (D-G) Eyes were then fixed and the 715

posterior sclera and retina removed to visualize the circumlental space (D&E) and how it 716

increased and decreased following treatment with either tropicamide (F) or pilocarpine (G). 717

(H) Summary of measurements taken from high power images showed that in control eyes (E), 718

the distance between ciliary processes and the lens was 151.51 ± 2.3 µm (mean ± SE). In eyes 719

treated with 0.2% tropicamide (J), the circumlental space was increased to 167.67 ± 1.4 µm 720

(mean ± SE). In eyes treated with 0.2% pilocarpine (I), the circumlental space was reduced to 721

129.03 ± 2.5 µm (mean ± SE). These differences were statistically significant (P < 0.05). 722

Statistical analysis was performed with a Mann-Whitney U test, P < 0.05 for each tested group. 723

724

725

726

727

728

729

730

731

732

733

734


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Figure 5. Effects of pharmacologically altering zonular tension on the subcellular 735

localization of AQP5 in the efflux zone of the rat lens. (A) Image montage of the water 736

efflux zone taken from a representative axial section of a rat lenses labelled with the membrane 737

marker WGA (red) and the nuclei marker DAPI (blue). The box indicates the area from which 738

high resolution images (B-D) were captured in lenses maintained in situ with their zonules 739

intact. (B-D) Top panels show nuclei, membrane and AQP5 labelling, while bottom panels 740

show only nuclei and AQP5 labelling from control lenses (B), and lenses incubated in 741

tropicamide (C), or pilocarpine (D) for 60 minutes. Note the shift from membranous labelling 742

to cytoplasmic labelling following incubation on pilocarpine. 743

744

745

746

747

748

749

750

751

752

753

754

755


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Figure 6. Subcellular localization of lens AQP0 and AQP5 in the influx zone of rat lenses 756

– results from the posterior pole. (A) Image montage of the posterior water influx zone taken 757

from an axial section of a rat lenses that was labelled with the membrane marker WGA (red) 758

to highlight suture line (arrow heads). Boxes indicate the areas (IP1, IP2 & IP3) from which 759

the higher resolution images shown in images B-D were taken from to investigate the 760

subcellular distribution of AQP0 and AQP5 (green). (B) In lenses with cut zonules AQP0 761

labelling was membranous and strongly labelled the suture in regions IP1 and IP2, but no 762

labelling was observed from region IP3 in the lens core where AQP0 the C-terminus of the 763

AQP0 protein is cleaved. (C) In lenses with zonules cut AQP5 labelling was missing from the 764

suture in regions IP1 and IP2, but labelling was present in the deeper IP3 region. (D) Fixing 765

lenses in situ with their zonules attached had no effect on AQP0 (data not shown) or AQP5 766

labelling in the posterior influx zone. 767

768

769

770

771

772

773

774

775

776

777


https://doi.org/10.1101/2020.06.29.178756


33

Figure 7. Subcellular localization of lens AQPs in the influx zone of rat lenses – results 778

from the anterior pole. (A) Image montage of the anterior water influx zone taken from an 779

axial section of a rat lenses that was labelled with the membrane marker WGA (red) to highlight 780

suture line (arrow heads). Boxes indicate the areas (IA1, IA2 & IA3) from which the higher 781

resolution images shown in images B-E were taken from to investigate the subcellular 782

distribution for each lens AQP (green). (B) In lenses with cut zonules AQP1 labelling was 783

present only in the epithelial cells of IA1 and was absent from IA2 and IA3. (C) In lenses with 784

cut zonules AQP0 labelling was membranous and strongly labelled the suture in regions IA1 785

and IA2, but no labelling was observed from region IA3 in the lens core where the C-terminus 786

of AQP0 protein is cleaved. (D) In lenses with zonules cut AQP5 labelling was missing from 787

the suture in regions IA1 and IA2, but labelling was present in the deeper IA3 region. (E) In 788

lenses that were fixed in situ with their zonules attached AQP5 labelling was still absent from 789

the suture in the peripheral region IA1, but was presence in regions IA2 and IA3. 790

791

792

793

794

795

796

797

798

799


https://doi.org/10.1101/2020.06.29.178756


34

Figure 8. Subcellular distribution of AQP5 in lenses with pharmacologically modulated 800

zonular tension at the anterior suture. (A) Image montage of the anterior water influx zone 801

taken from an axial section of a rat lenses that was labelled with the membrane marker WGA 802

(red) to highlight suture line (arrow heads). Boxes indicate the areas (IA1, IA2 & IA3) from 803

which the higher resolution images shown in images B&C were taken from to investigate the 804

subcellular distribution of AQP5 (green) following the application of tropicamide (B) or 805

pilocarpine for 60 minutes. (B) Application of tropicamide did not change AQP5 labelling in 806

the suture (compare to Figure 6E). (C) In lenses treated with pilocarpine AQP5 labelling was 807

absent from the sutures of regions IA1 and IA2, but was still present at IA3. 808

809

810

811

812

813

814

815

816

817

818

819

820


https://doi.org/10.1101/2020.06.29.178756


35

Figure 9. Schematic representation of the subcellular localization of AQP5 in the efflux 821

and influx zones of the rat lens in presence and absence of zonular tension. (A) Epithelial 822

cells (dark blue) which differentiate into fiber cells (blue) in the equatorial efflux zone (E1) are 823

initially attached by their apical membrane domains to form the modiolus. As fiber cells detach 824

from the modiolus their apical and basal tips migrate along the epithelium and capsule, 825

respectively, and their lateral membranes undergo massive elongation. This process of 826

elongation continues until the apical and basal tips of fiber cells (light blue) from opposing lens 827

hemisphere meet to form the anterior and posterior sutures, respectively. As this process 828

continues throughout life newly differentiated secondary fiber cells internalize older mature 829

fiber cells which in turn internalized the primary fiber cells (yellow) laid down during 830

embryonic development. Boxes represent the regions in the efflux and influx zones where the 831

subcellular localization of AQP5 was measured. (B) Schematic representation of a fiber cell 832

depicting its three specific membrane domains consisting of the apical tip, lateral membranes 833

and basal tip. (C) Table summarizing the regional differences of AQP5 subcellular localization 834

observed in the efflux and influx zones in the presence and absence of zonular tension. 835

836

837

838

839


https://doi.org/10.1101/2020.06.29.178756


36

840

Figure 1. Structure and physiology of the lens. 841

842


https://doi.org/10.1101/2020.06.29.178756


37

843

Figure 2. Subcellular localization of AQP1, 0 and 5 at the efflux zone of water movement 844

in the outer cortex of immediately fixed rat lenses with their zonules cut. 845

846


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38

847

848

Figure 3. Subcellular localization of AQP5 at the efflux region of movement of water in 849

time series incubation of organ cultured rat lenses with mechanically manipulated change 850

of zonular tension. 851

852


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39

.853

854

Figure 4. Tropicamide and pilocarpine pharmacological agents modulate the zonular 855

tension of the rat lens. 856

857


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40

858

Figure 5. Modulation of the zonular tension with the pharmacological agents tropicamide 859

and pilocarpine change the subcellular localization of AQP5 at the efflux region of 860

movement of water. 861

862


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41

863

Figure 6. Subcellular localization of AQP0 and AQP5 in presence and absence of zonular 864

tension at the posterior suture. (Scale bars) 865

866


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42

867

Figure 7. Subcellular localization of AQP1, 0 and 5 in presence and absence of zonular 868

tension at the anterior suture. 869

870


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43

871

872

Figure 8. Subcellular distribution of AQP5 in lenses with pharmacologically modulated 873

zonular tension at the anterior suture. 874

875


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44

876

877

Figure 9. Schematic representation of the subcellular localization of AQP5 in the efflux 878

and influx zones of the rat lens in presence and absence of zonular tension. 879

880


https://doi.org/10.1101/2020.06.29.178756

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Petrova et al Dynamic changes in AQP5 membrane localization · 29/06/2020 · Petrova et al...

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