Hypotonicity promotes epithelial gap closure by lamellipodial protrusion

Accepted Manuscript Hypotonicity promotes epithelial gap closure by lamellipodial protrusion T. Chen, H. Zhao, L. Gao, L. Song, F. Yang, J. Du PII:

S0079-6107(17)30187-6

DOI:

10.1016/j.pbiomolbio.2017.09.021

Reference:

JPBM 1284

To appear in:

Progress in Biophysics and Molecular Biology

Received Date: 31 July 2017 Revised Date:

11 September 2017

Accepted Date: 25 September 2017

Please cite this article as: Chen, T., Zhao, H., Gao, L., Song, L., Yang, F., Du, J., Hypotonicity promotes epithelial gap closure by lamellipodial protrusion, Progress in Biophysics and Molecular Biology (2017), doi: 10.1016/j.pbiomolbio.2017.09.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Running title: Hypotonicity promotes gap closure

Hypotonicity promotes epithelial gap closure by lamellipodial protrusion

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T. Chen1,3, H. Zhao1, L. Gao4, L. Song1,4, F. Yang3, and J. Du1,2* Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, Beijing

100084, People’s Republic of China

Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and

Medical Engineering, Beihang University, Beijing 100191, China

Department of Orthopaedics, Trauma and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University,

Guangxi 530027, People’s Republic of China 4

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College of life science, Hebei normal university, Heibei 050024, People’s Republic of China

ABSTRACT

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*Corresponding Authors: Jing Du, E-mail: [email protected]

The closure of gaps within epithelia is an essential part of many physiological and pathological

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processes, such as embryonic development, organ remodeling and wound healing. Emerging evidence

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proved that the physical microenvironment plays important roles in cell behaviors. However, the effect of osmolarity of extracellular medium on gap closure is least understood. Using a gap closure model of epithelial cells, we found that hypotonic condition significantly facilitated the process of gap closure. Moreover, instead of actomyosin ring, enhanced migration leading by lamellipodia primarily contributed to the rapid gap closure in hypotonic condition. These findings provide insights for understanding the physiology of epithelial gap closure.

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Key words: gap closure, hypotonicity, lamellipodial

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INTRODUCTION

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Rapid epithelial gap closure is an essential biological process for tissue homeostasis maintenance during many physiological and pathological processes such as embryonic morphogenesis and wound healing. In embryogenesis, it requires collective cell migration to close the gap, including D.

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melanogaster dorsal closure, C. elegans ventral enclosure, eyelid closure, neural tube closure and trachea invagination (Brugues et al., 2014; Hashimoto et al., 2015; Heller et al., 2014; Martin and

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Parkhurst, 2004). In wound healing, it can maintain the tissue homeostasis so that it restricts exposure of the inside of an organism to the noxious outside environment (Crosby and Waters, 2010; Kuipers et al., 2014).

There are two main mechanisms commonly accepted in gap closure (Bement et al., 1999; Fenteany

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et al., 2000; Poujade et al., 2007; Tamada et al., 2007). The first mechanism, referred to as actin pursestring contraction, is mediated by the coordinated contraction of actin bundles along the wound circumference (Bement et al., 1993; Nobes and Hall, 1995). The second mechanism, termed cell

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crawling, is the collective migration of marginal and submarginal cells led by lamellipodial protrusion (Kim et al., 2010; Salbreux et al., 2009). In most case, the two mechanisms are both present. In only a

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few cases, one mechanism dominates (Abreu-Blanco et al., 2012; Ravasio et al., 2015). For instance, epithelial gap can be closed only via purse-string occuring over non-adherent surfaces (Vedula et al., 2015). Besides, there are other factors to regulate the mechanism of gap closure including gap geometry, gap size, ECM coating, cell type, and substrate stiffness (Anon et al., 2012; Klarlund, 2012). These factors may affect one mechanism or both of the two mechanisms. In many instances, epithelial gap closure occurs in a hypotonic medium. Because epithelia can maintain large ionic concentration differences between the extracellular space and the external 03

ACCEPTED MANUSCRIPT Running title: Hypotonicity promotes gap closure environment (Gilljam et al., 1989; Joris et al., 1993). For example, the epithelial cells of mouth and esophagus can protect the inside tonicity of the tissues (~270–300 mOsm, i.e., common vertebrate extracellular tonicity) from hypotonic saliva (~30 mOsm) (Enyedi et al., 2013).

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The classic scratch wound assay is the most commonly methods to creat gap within cell monolayers (Todaro et al., 1965). This method is simple, economy and time-saving. However, it is difficult to create identical gap. During the process of scratch, it may release death factors and cell debris that

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disrupt the gap closure (Block et al., 2004; Klepeis et al., 2001). To overcome these drawbacks, we used poly-dimethylsioxane (PDMS) micropillars to creat well-defined cell gap (Anon et al., 2012). By

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using a stencil of PDMS micropillars, circular gaps with 180 µm diameter were obtained. The PDMS pillars were coated with pluronics to prevent cell attachment and make cells adhere to the substrate surrounding the pillars. Madin-Darby canine kidney (MDCK) cells were cultured in between the pillars for 18 ± 3 h. Upon careful removal of the PDMS pillar, a gap was created within the monolayer (Anon

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et al., 2012). Then, we used this cell model to study the dynamics of gap closure in isotonic media (320 mOsm) and hypotonic media (160 mOsm), respectively (Gauthier et al., 2011; Mao et al., 2005; Stroka et al., 2014).

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In this paper, we compared the dynamics of gap closure under normal tonicity and hypotonic medium. We found that the gap closure was more quickly completed in hypotonic condition, and the

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mechanism involved lamellipodial protrusion-leading cell migration.

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MATERIALS AND METHODS

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Cell culture and hypotonic media MDCK Cells were cultured in DMEM medium (containing with 4.5 g/L glucose, L-glutamine, and sodium pyruvate) supplemented with 10% FBS (Life technologies, CA, USA), and 100 IU/mg

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penicillin–streptomycin (Life technologies, CA, USA) at 37°C and 5% CO2. Isotonic medium (320mOsm) is DMEM with 10%FBS, hypotonic medium (160mOsm) was created by mixing DMEM

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(with 10%FBS) with deionized, filtered water. Osmotic pressure was measure by using an osmometer.

Drug treatments

Pharmacological inhibitors [Y27632 (Sigma), NSC23766 (Sigma)] were perfused at least 2 h before

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releasing the PDMS stamp.Their concentrations both are 20 µM.

Preparation of PDMS stencils

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The fabrication of the PDMS stencils was based on a previously described(Anon et al., 2012; Ravasio et al., 2015). Briefly, shapes and geometries which we need were transferred from a mask on a silicon

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wafer by photolithography. By using deep reactive ion etching technique, we create pillars (height ~100µm) emerging from the wafer. After silanization of the wafer by vapour deposition (1H, 1H, 2H, 2H-Perfluorooctyl-trichlorosilane; Sigma), PDMS at a ratio 1/10 (curing agent/base) was poured over the silicon template and cured at 70°C for 8h. Then, we can peeled off PDMS pillars from the silicon template. Preparation of wound-free gaps in the MDCK-confluent epithelium. The methods was based on a previously described(Anon et al., 2012; Ravasio et al., 2015).Briefly, 20µg/ml fibronectin solution was 05

ACCEPTED MANUSCRIPT Running title: Hypotonicity promotes gap closure incubated for 1 h at 37°C on a glass-bottom Petri dish (Corning). The sample was then submerged for 1 h in a 0.2% pluronic acid solution (Sigma) to prevent cell adhesion on the pillar walls. Cells were seeded highly concentrated in a small volume (15µl with~100,000 cells), close to the pillar stamp, to

formed, and PDMS stencils were carefully peeled off with tweezers.

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Video Microscopy and Image Analysis

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allow an even distribution of the cells between the pillars. After 15 ± 3 h a confluent monolayer was

Live cell imaging was performed in Olympus microscope, enclosed in an incubator to maintain the

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samples at 37 °C and 5% of CO2 throughout the experiments. Images were acquired every 5min with Nikon software. Images were analyzed by software ImageJ.

Fluorescent Staining

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After careful removal of the stencils, samples were washed with PBS and fixed with hypotonic /isotonic media in the incubator. Then, cells were rinsed with PBS and fixed with 4% paraformaldehyde(PFA) for 30 min at room temperature. After fixation, the cells were rinsed three

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times with PBS and permeabilized for 10 min with PBS/0.2% Triton X-100, washed three times with PBS. Then, the cells blocked for 1 h with 5% BSA in PBS at 37°C, incubated with Alexa Fluor 488

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Phalloidin (Life technologies, CA, USA) in PBS for 1 h at room temperature. Images were taken at 63X or 40X magnification by Leica TCS-SP5 Confocal Microscope (Leica Microsystems, Wetzlar, Germany). The integral optical density (IOD) and cell plasma or nuclear areas of immunofluorescence pictures were counted by the tool of Imagepro Plus.

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RESULTS We first analyzed the dynamics of epithelial gap closure in different osmotic pressure after removal of

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pillars. We tracked the perimeter of the gap every 5 minutes (Fig. 1a and b; Movie S1 and 2). The plots show that the closure was linear and occasionally fluctuated. In the hypotonic condition, the average velocity of complete closure was 3.93 ± 1.04 µm/min (n = 5, mean ± s.d.), while in the isotonic

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condition, the velocity was 1.76 ± 0.18 µm/min (n = 6, mean ± s.d.) (Fig. 1c). These results suggest that hypotonic condition significantly promoted epithelial gap closure (Fig. 1d).

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To explore the mechanism underlying promoted gap closure by hypotonicity, we analyzed the lamellipodia and actomyosin ring formation, respectively. According to the time-lapse microscopy results, cells at the gap broader extended lamellipodia throughout the process of closure. Evident lamellipodia was observed after the release of the pillar (Fig. 1a and b). We tracked the area of the

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lamellipodia every 5 minutes. The formation of lamellipodia was random and could be observed both in hypotonic and isotonic conditions. However, compared with isotonic condition, low osmolarity significantly facilitated lamellipodia protrusion, indicated by the higher occurence frequency and larger

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area of lammellipodia in hypotonic condition (Fig. 2).

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Running title: Hypotonicity promotes gap closure

Figure 1 Hypotonicity promoted gap closure. Phase contrast images during gap closure in isotonic (a)

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and hypotonic (b) conditions. Triangles show cells forming lamellipodia. The area enclosed by a blue line is lamellipodia in isotonic condition, while the area enclosed by a yellow line is lamellipodia in

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hypotonic condition. (c) Dynamics of gap closure in isotonic or hypotonic condition. (d) Closure time of the different osmotic pressure. Error bars indicate s.d. Samples are considered statistically different for P<0.05 in unpaired Student’s t-test and are indicated by the star. (Scale bars, 20 µm)

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Figure 2 Extensive lamellipodia formation in hypotonic condition. (a) The dynamics of lamellipodial area during gap closure in isotonic or hypotonic medium were monitored by time-lapse microscope. (b) Lamellipodial mean area of the different osmotic pressure. Error bars indicate s.d. Samples are

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considered statistically different for P<0.05 in unpaired Student’s t-test and are indicated by the star.

Next, we investigated F-actin distribution at the gap edge. Phalloidin staining 15 minutes after pillar

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removal showed that F-actin accumulated in a continuous supracellular cable-like structure at the

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margins of the gap in isotonic condition (Fig. 3a). However, in hypotonic condition, no actin clustering at the gap interface was observed (Fig. 3c). Peak of F-actin intensity demonstrated an enrichment of Factin at gap border (higher and narrower) in isotonic condition, compared with hypotonic condition (Fig. 3b and d). Moreover, in isotonic condition, the mean intensity ratio of gap margrins to 5 µm outside the border was higher than hypotonic condition (Fig. 3e). However, consistent with Figure 2, significant lamellipodia formation was observed at the advancing cell front in hypotonic medium (Fig. 3c). 09

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Figure 3 Distinct F-actin distribution in isotonic and hypotonic conditions. The assembly of F-actin

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was stained by phalloidin in isotonic condition (a) and hypotonic condition (b). Quantification of Factin fluorescence intensity in radial direction in isotonic condition (c) and hypotonic condition (d). (e) The illustration figure of the definition of the mean intensity ratio at gap margrins. (f) The mean

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intensity ratio of gap margrins to 5 µm outside the border in different osmotic pressure. The higher ratio is the more intensive F-actin accumulated at the border. Triangles show cells forming

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lamellipodia. Error bars indicate s.d. Samples are considered statistically different for P<0.05 in unpaired Student’s t-test and are indicated by the star (n = 6). (Scale bars, 40 µm)

Furthermore, we evaluated the function of lamellipodia extension and actomyosin ring contractility in hypotonicity promoted gap closure. Specific inhibitor of ROCK or Rac1 was added to the cell medium during gap closure to inhibit actomyosin ring or lamellipodia, respectively (Nobes and Hall, 010

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indicate that extensive lamellipodia formation may contribute to the facilitated gap closure by

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hypotonicity.

Figure 4 Mechanism of gap closure: closure time of the different osmotic pressure in control conditions and subjected to Rac1 inhibition and ROCK inhibition. Error bars indicate s.d. Samples are

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considered statistically different for P<0.05 in unpaired Student’s t-test and are indicated by the star. (n

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= 4)

DISCUSSION

Osmotic stress has a primary impact on the the cell migration. In hypotonic medium, an increase in cell tension reduces lateral membrane protrusions in the lamellipodium, composed of longer actin filaments oriented toward the direction of movement (Batchelder et al., 2011). Thus, evidence suggests

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ACCEPTED MANUSCRIPT Running title: Hypotonicity promotes gap closure that hypotonicity promotes single cell migration. However, the mechanisms of collective cell migration in hypotonicity are still unknown. In this study, we found that hypotonicity promoted gap closure probably through enhanced

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lamellipodial formation. Without actin cable, gap closure is more quickly in hypotonic condition, which indicates cell crawling appears to be the dominant mechanism of gap closure. Consistently, Philipp Niethammer et al. found that hypotonicity accelerated zebrafish fin healing (Gault et al., 2014).

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They found that osmolarity differences between interstitial fluid and the external environment mediate rapid leukocyte recruitment to sites of tissue damage in zebrafish by activating cytosolic phospholipase

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a2 (cPLA2) at injury sites (Enyedi et al., 2013). As is well known, wound repair is still considered as a process driven by cell damage, lack of contact inhibition, or altered mechanical signaling at tissue edges (Block et al., 2004; Fagotto and Gumbiner, 1996; Jacinto et al., 2001; Martin, 1997; Zegers et al., 2003). Therefore, our findings demonstrate the importance of osmotic regulation in gap closure and

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thus provide new mechanistic insights into this process.

AUTHOR CONTRIBUTIONS

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J. D., F. Y., and H. Z. conceived the study, designed the experiments, and wrote the manuscript. T.

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C., L. G., and L. S. performed the experiments and analyzed the data.

ACKNOWLEDGEMENTS

This work was supported by the National Key R&D Program of China (2017YFA0506500) and the National Natural Science Foundation of China (Grants No. 31370018). The authors declare no competing financial interests.

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