Cadherin-6B undergoes macropinocytosis and clathrin-mediated endocytosis during cranial neural crest cell EMT.

© 2015. Published by The Company of Biologists Ltd.

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Cadherin-6B undergoes macropinocytosis and clathrin-mediated endocytosis during

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cranial neural crest cell EMT

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Rangarajan Padmanabhan†, Lisa A. Taneyhill#*

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Journal of Cell Science

Accepted manuscript

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# Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742 † National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

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*Corresponding author

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Address for manuscript correspondence:

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e-mail: [email protected]

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Tel: 301 405 0597

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Fax: 301 405 7980

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Running Title: Cadherin-6B endocytosis and macropinocytosis in the neural crest

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Keywords: Cadherins, EMT, neural crest, macropinocytosis, endocytosis

JCS Advance Online Article. Posted on 20 March 2015

Accepted manuscript

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Abstract

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The epithelial-to-mesenchymal transition (EMT) is critical for the formation of migratory neural

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crest cells during development and is co-opted in human diseases such as cancer metastasis.

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Chick premigratory cranial neural crest cells lose intercellular contacts, mediated in part by

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Cadherin-6B (Cad6B), migrate extensively, and later form a variety of adult derivatives.

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Importantly, modulation of Cad6B is critical for proper neural crest cell EMT. Although Cad6B

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possesses a long half-life, it is rapidly lost from premigratory neural crest cell membranes,

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suggesting the existence of post-translational mechanisms during EMT. We have identified a

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motif in the Cad6B cytoplasmic tail that enhances Cad6B internalization and reduces the stability

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of Cad6B upon its mutation. Furthermore, we demonstrate for the first time that Cad6B is

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removed from premigratory neural crest cells through cell surface internalization events that

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include clathrin-mediated endocytosis and macropinocytosis. Both of these processes are

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dependent upon the function of dynamin, and inhibition of Cad6B internalization abrogates

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neural crest cell EMT and migration. Collectively, our findings reveal the significance of post-

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translational events in controlling cadherins during neural crest cell EMT and migration.


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Neural crest cells are a multipotent cell population arising at the border of the neural and non-

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neural ectoderm. Initially immotile in the dorsal region of the chick neural tube and termed

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premigratory neural crest cells, these cells undergo an epithelial-to-mesenchymal transition

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(EMT) to emerge from the neural tube. Migratory neural crest cells later differentiate into a

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variety of specialized adult derivatives such as the neurons and glia of peripheral nervous

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system, craniofacial tissues, portions of the heart, and melanocytes (Hall, 2009; Dupin, 2014).

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EMT plays a central role not only during normal embryonic development and adult homeostasis, but also in pathological conditions such as cancer metastasis and fibrosis (Thiery et al., 2009;

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Nieto, 2011), and requires a decrease in cell-cell adhesion, loss of apicobasal polarity, up-

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regulation of mesenchymal markers, and finally initiation of migration (Hay, 1995; Nieto, 2011).

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As such, neural crest cells are a popular model to study EMT because they provide a relevant, in

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vivo system in which to examine molecular mechanisms underlying EMT and migration directly

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translatable to aberrant EMTs occurring during human disease (Hay, 1995; Theveneau and

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Mayor, 2012; Kulesa et al., 2013).

Accepted manuscript

INTRODUCTION

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Chick premigratory cranial neural crest cells express several cell adhesion molecules,

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including those comprising adherens and tight junctions (Nakagawa and Takeichi, 1995; Coles et

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al., 2007; Wu et al., 2011; Dady et al., 2012; Fishwick et al., 2012). Many of these proteins are

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undetectable upon initiation of EMT and early migration, suggesting that their down-regulation

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is important (Nakagawa and Takeichi, 1995; Coles et al., 2007; Wu et al., 2011; Dady et al.,

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2012; Fishwick et al., 2012). Cadherins are central components of adherens junctions, and, along

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with nectin/afadins, form the “adhesion belt” through interactions with circumferential F-actin,

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linking cells into a continuous sheet and separating the apical and basolateral membranes

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(Farquhar and Palade, 1963; Takai et al., 2008; Meng and Takeichi, 2009). Chick premigratory

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cranial neural crest cells express at least three cadherins: Cadherin-6B (Cad6B), N-cadherin, and

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E-cadherin (Hatta and Takeichi, 1986; Duband et al., 1988; Nakagawa and Takeichi, 1995;

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Nakagawa and Takeichi, 1998; Dady et al., 2012). Expression of E-cadherin is high in

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prospective neural crest cells prior to neurulation, but as neurulation progresses, E-cadherin is

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gradually reduced and only retained until early stages of neural crest cell delamination. N-

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cadherin protein, however, is expressed during neurulation but is lost before EMT in

Accepted manuscript Journal of Cell Science

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premigratory cranial neural crest cells (Dady et al., 2012; Rogers et al., 2013). In contrast to E-

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cadherin, Cad6B is uniquely restricted to the premigratory cranial neural crest cell population.

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Cad6B protein is observed in the neural folds, gradually increases as premigratory neural crest

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cells prepare for EMT, and is completely down-regulated as neural crest cells undergo EMT and

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migrate (Nakagawa and Takeichi, 1995; Nakagawa and Takeichi, 1998; Taneyhill, 2008). A

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reduction in Cad6B is critical for the emergence of cranial neural crest cells from the neural tube,

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as Cad6B overexpression or knock-down inhibits or enhances this process, respectively (Coles et

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al., 2007).

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Cadherins are removed from cellular plasma membranes during EMT through multiple

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post-translational mechanisms, including proteolytic processing and endocytosis (McCusker and

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Alfandari, 2009; Ulrich and Heisenberg, 2009; Kowalczyk and Nanes, 2012). Upon endocytosis,

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cadherins are either recycled back to the plasma membrane (Le et al., 1999; Classen et al., 2005;

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Desclozeaux et al., 2008) or degraded in lysosomes (Xiao et al., 2003b; Palacios et al., 2005).

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Cadherins can be internalized through clathrin-dependent and -independent endocytosis (Le et

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al., 1999; Akhtar and Hotchin, 2001; Paterson et al., 2003; Bryant et al., 2005; Palacios et al.,

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2005; Xiao et al., 2005; Bryant et al., 2007; Toyoshima et al., 2007). Indeed, the cytoplasmic

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domain of several cadherins harbors motifs that have been demonstrated to regulate clathrin-

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mediated endocytosis (Miyashita and Ozawa, 2007b; Chiasson et al., 2009; Ishiyama et al., 2010;

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Nanes et al., 2012). In addition to endocytosis, macropinocytosis, in which whole adherens

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junctions are internalized, also regulates cell surface cadherin levels (Paterson et al., 2003;

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Bryant et al., 2007; Sharma and Henderson, 2007; Solis et al., 2012). Furthermore, both clathrin-

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mediated endocytosis and macropinocytosis can rely upon dynamin for vesicle scission from the

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plasma membrane (Jarrett et al., 2002; Orth et al., 2002; Palacios et al., 2002; Cao et al., 2007).

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We recently showed that ADAM-mediated proteolysis of Cad6B is critical to remove

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Cad6B protein from the plasma membrane of premigratory cranial neural crest cells and

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facilitate EMT (Schiffmacher et al., 2014). In this study, we explored whether endocytosis plays

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an additional role during cranial neural crest cell EMT. We now show for the first time that

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premigratory cranial neural crest cells internalize Cad6B during EMT through clathrin-mediated

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endocytosis and macropinocytosis, that latter of which likely involves removal of whole

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adherens junctions from the plasma membrane. Furthermore, both of these processes depend

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upon the function of dynamin, and loss of Cad6B internalization prevents neural crest cell EMT

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and migration. Taken together, our results highlight a crucial role for cadherin internalization

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during an in vivo EMT in cranial neural crest cells.

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RESULTS

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Cad6B undergoes internalization and localizes to the cytoplasm in vitro, in vivo, and ex vivo

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To explore the possibility that Cad6B might undergo internalization via endocytosis, FlpInCHO-

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Cad6B cell lines were generated that stably express wild-type Cad6B (tagged with a

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hemagglutinin (HA) epitope at the C-terminus) from a single genomic locus (FlpIn-wtC6B)

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(Schiffmacher et al., 2014). This approach was taken to minimize any deleterious effects that

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might occur due to Cad6B overexpression (Levenberg et al., 1999; Bryant et al., 2005), and CHO

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cells were chosen because they do not express any cadherins (Hong et al., 2010). We first

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determined whether Cad6B undergoes basal levels of internalization in these cells by performing

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indirect immunofluorescence using antibodies that recognize the N terminal, extracellular


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domain of Cad6B (NT-6B) and Na+K+ATPase, a marker of the plasma membrane (Van Dyke,

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2004) (other antibodies raised to proteins marking the endocytic/lysosomal compartments did not

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work on chick tissue, data not shown). Since Na+K+ATPase plays an important role in the

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acidification of endosomes (Cain et al., 1989; Fuchs et al., 1989; Feldmann et al., 2007) and

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localizes to Rab5-positive endosomes (Feldmann et al., 2007), we reasoned that cytoplasm-

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localized Na+K+ATPase could serve as a marker for early endosomes. Through confocal

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microscopy analysis of Z-stack images, we observe Cad6B on the plasma membrane and in the

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cytoplasm in apparent puncta, where it co-localizes with Na+K+ATPase (Fig. 1A-A”,

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arrowheads). These results suggest that Cad6B undergoes internalization and localizes to the

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cytoplasm in this cell line. To ascertain whether internalization plays a role in the post-

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translational down-regulation of Cad6B in vivo, we performed immunohistochemistry for Cad6B

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and Na+K+ATPase on 6 somite stage (ss) and 7ss embryos, when premigratory cranial neural

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crest cells begin to reduce Cad6B levels during EMT (Taneyhill et al., 2007; Schiffmacher et al.,

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2014). In addition to Cad6B marking cell membranes (Fig. 1B-B’’, arrow), we observe Cad6B in

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cytoplasmic puncta co-localizing with Na+K+ATPase in premigratory cranial neural crest cells

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(Fig 1B”, arrowheads), pointing to the possibility that Cad6B is internalized in these precursors

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explant assays (Taneyhill et al., 2007) and examined Cad6B in neural crest cells undergoing

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EMT ex vivo in culture. Immunostaining for Cad6B reveals several puncta in the cytosol of

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emerging neural crest cells, corroborating our in vivo and in vitro observations (Fig. 1C-C’,

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arrowheads).

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To determine if the cytoplasmic puncta observed in vivo were of endocytic or exocytic

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nature, we performed Cad6B antibody feeding assays (Arancibia-Carcamo IL, 2006) in which

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we introduced the NT-6B antibody into the lumen of embryos electroporated with a membrane

Accepted manuscript

prior to and/or during EMT. To address this further, we conducted cranial neural crest cell

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GFP construct (to label premigratory neural crest cell plasma membranes). Since the NT-6B

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antibody recognizes the lumen-exposed, extracellular domain of Cad6B, the Cad6B antibody-

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antigen complex should be processed in the same way as endogenous Cad6B protein when

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premigratory cranial neural crest cells undergo EMT. Through confocal imaging, we note Cad6B

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in several cytosolic puncta (Fig. 2B”, arrowheads) within the confines of a membrane GFP-

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expressing delaminating neural crest cell (Fig. 2B”, arrow), implying that the puncta are likely to

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be of endocytic rather than exocytic nature. To eliminate the possibility that the binding of the


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NT-6B antibody was stimulating endocytosis, we performed the same antibody assay in vitro

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(Fig. 2C-C”) and ex vivo (Fig. 2D-D”). The plasma membrane-bound Cad6B antibody-antigen

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complex (un-internalized fraction) was distinguished from the internalized fraction by washing

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the cells with a low pH buffer, which strips off any plasma membrane-bound antibodies but does

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not disturb the internalized pool (Arancibia-Carcamo IL, 2006). Plasma membrane-bound Cad6B

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was distinguished from the internalized Cad6B antibody-antigen complex by performing

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immunofluorescence for the HA tag at the C-terminal end of Cad6B. When the assay was

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performed at 4˚C, most of the Cad6B antibody was stripped off due to the continued presence of

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Cad6B on the membrane (Fig. S1A) without affecting membrane distribution of Cad6B, as

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shown by the HA immunoreactivity (Fig. S1A”, arrows). Performing the assay at 37˚C resulted

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in the presence of several internalized NT-6B-Cad6B cytoplasmic plasma (Fig. 2C”,

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arrowheads), but did not stimulate global endocytosis of Cad6B protein at 37˚C, as indicated by

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the presence of retained HA (Cad6B) immunoreactivity on the plasma membrane (Fig. 2C”,

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arrows). The same assay was then performed with explants (Fig. S1B-B” and 2D-D”). Here the

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plasma membrane-bound fraction of Cad6B was differentiated from the endocytosed Cad6B

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antibody-antigen complex with an antibody that recognizes the C-terminal domain of


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endogenous Cad6B (CT-6B). Explants remaining at 4˚C that were subjected to the low pH wash

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had virtually all Cad6B antibody bound to plasma membrane-bound Cad6B removed without

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affecting general Cad6B membrane distribution (Fig. S1B’, B”, arrows). Incubating the explants

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at 37˚C, however, led to the presence of several NT-6B-Cad6B cytosolic puncta (Fig. 2D”,

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arrowheads) and the persistence of some membrane-bound Cad6B (Fig. 2D”, arrows). These

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data suggest that addition of the NT-6B antibody does not stimulate significant internalization of

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Cad6B and corroborate our earlier observation that the puncta are likely endocytic rather than

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exocytic in nature. Taken together, these observations reveal that Cad6B undergoes

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internalization in cultured cells and in premigratory cranial neural crest cells in vivo and ex vivo.

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Cad6B possesses a functional endocytic motif in its cytoplasmic domain

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Several cadherins possess motifs within their C terminal intracellular domains that regulate their

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internalization and degradation (Xiao et al., 2005; Miyashita and Ozawa, 2007a; Miyashita and

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Ozawa, 2007b; Tai et al., 2007; Hong et al., 2010; Kowalczyk and Nanes, 2012; Nanes et al.,

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2012). We compared the amino acid sequence of the cytoplasmic domain of Cad6B to that of

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several

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(http://www.ebi.ac.uk/Tools/msa/clustalw2/), and in doing so identified two motifs that could

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potentially regulate Cad6B internalization (Fig. 3A, yellow highlight). Mutation of the dileucine

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motif has been shown to negatively influence endocytosis (Miyashita and Ozawa, 2007b; Hong

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et al., 2010), whereas changes in the p120-catenin binding motif can promote endocytosis due to

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the inability of p120-catenin to bind and prevent endocytosis (Ireton et al., 2002; Xiao et al.,

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2003a; Xiao et al., 2005; Miyashita and Ozawa, 2007b; Ishiyama et al., 2010; Nanes et al.,

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2012). To investigate the function of these two motifs, we performed site-directed mutagenesis

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to create two Cad6B mutant constructs, LI645AA and EED666AAA, and expressed each

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independently from a single genomic locus in the FlpIn-CHO cell lines as described earlier. We

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hypothesized that mutating the LI or EED residues would decrease or augment endocytosis,

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respectively. Immunofluorescence analysis with the NT-6B antibody qualitatively revealed that,

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compared to wild-type Cad6B (Fig. 3B-B”, arrows) and the LI645AA mutant (Fig. 3C-C”,

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arrows), the EED666AAA mutant localized predominantly to the cytoplasm, indicating that

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these residues might modulate Cad6B distribution (Fig. 3D-D”, carets). Furthermore, wild-type

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Cad6B and the LI mutant co-localize with p120-catenin at the cell membrane (Fig. 3B”, C”,

type

II

cadherins

through

the

ClustalW

algorithm

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mutant. Finally, we note intracellular puncta that are Cad6B-positive but p120-catenin-negative

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(Fig. 3B”, D”, carets).

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To evaluate the ability of these cells to form stable cytoskeletal-associated cell-cell

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junctions, we investigated the proportion of TX100-soluble and -insoluble Cad6B (Fig. 4A, B).

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We observed a statistically significant reduction in the proportion of actin-anchored Cad6B in

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the EED666AA mutant (p < 0.05), suggesting that these cells are deficient in forming stable cell-

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cell adhesions. We then quantified the steady state level of internalization of wild-type Cad6B

Accepted manuscript

arrows), whereas p120-catenin is observed diffusely throughout the cytoplasm in the EED

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and the mutants through biotinylation assays. Although wtCad6B and LI645AA do not show

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significant differences in endocytosis, the EED666AAA mutant undergoes rapid endocytosis by

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60 minutes post-biotinylation (Fig. 4C, D, p < 0.05). Surprisingly, the levels of endocytosed

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Cad6B in the EED666AAA mutant decreased at 180 minutes post-biotinylation. We reasoned

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that the absence of these residues might negatively impact Cad6B stability, making it more

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susceptible to degradation and resulting in decreased levels of endocytosed protein at 180

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minutes. To investigate this possibility, we inhibited de novo protein synthesis through


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cycloheximide treatment of wild-type Cad6B and mutant cells and analyzed levels of Cad6B.

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Compared to wild-type Cad6B and LI645AA, the EED666AAA mutant undergoes rapid

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reduction in Cad6B, substantiating our hypothesis (Fig. 4E, F, p < 0.001). Collectively, these

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observations reveal that Cad6B undergoes endocytosis and possesses a functional motif that

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negatively regulates its endocytosis in vitro.

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In vitro and in vivo internalization of Cad6B is mediated, in part, through clathrin-

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dependent endocytosis

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Our biochemical and immunostaining data suggest that Cad6B is internalized through

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endocytosis. Cadherins can be internalized through both clathrin-dependent and -independent

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endocytic pathways (Le et al., 1999; Akhtar and Hotchin, 2001; Paterson et al., 2003; Bryant et

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al., 2005; Palacios et al., 2005; Xiao et al., 2005; Bryant et al., 2007; Toyoshima et al., 2007). To

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determine through which pathway Cad6B is endocytosed, we performed immunohistochemistry

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to document the distribution of Cad6B with respect to caveolin-1 and clathrin. We note that

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intracellular Cad6B does not co-localize with caveolin-1 (Fig. 5A-A”, carets) but partially co-

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localizes with clathrin in vitro (Fig. 5B-B”, arrowheads). This co-localization was corroborated


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by examining Cad6B and clathrin sub-cellular distribution in the LI645AA (Fig. 5C-C”,

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arrowheads) and EED666AAA (Fig. 5D-D”, arrowheads) mutant cell lines. Intriguingly, though,

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all cell lines show some intracellular puncta that are Cad6B-positive but devoid of clathrin

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immunoreactivity (Fig. 5B”, C”, D”, carets).

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To confirm these findings in vivo, embryos at stages where premigratory cranial neural

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crest cells are undergoing EMT were immunostained for Cad6B and clathrin (Fig. 6). As

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observed in vitro, Cad6B co-localizes with clathrin in vivo (Fig. 6B-B”, arrowheads). Once

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again, though, we note some Cad6B-positive puncta that lack clathrin (Fig. 6B”, carets). Taken

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together, these data indicate that Cad6B undergoes clathrin-mediated endocytosis in vitro and in

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vivo during EMT. The presence of clathrin-negative, Cad6B-positive puncta, however, is

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suggestive of additional mechanism(s) by which Cad6B could be internalized.

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Cad6B undergoes dynamin-dependent, clathrin-mediated endocytosis and

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macropinocytosis in neural crest cells undergoing EMT

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Our cell line data revealed that Cad6B co-localizes with p120-catenin in some intracellular

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puncta (Fig. 3B”, C”, D”, arrowheads). To corroborate this in vivo, we performed

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immunohistochemistry for Cad6B and p120-catenin on chick cranial transverse sections. Our

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results show that Cad6B and p120-catenin co-localize to cytoplasmic puncta/vesicles and at the

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plasma membrane (Fig. 7, arrowheads, arrow, respectively; p120-catenin-negative, Cad6B-

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positive puncta are indicated by carets). Endocytosis of cadherins, however, relies upon the

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removal of the p120-catenin protein (Davis et al., 2003; Xiao et al., 2003a; Hoshino et al., 2005).

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This co-localization data suggest that, in some instances, whole complexes containing Cad6B

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and catenins, rather than individual Cad6B molecules, could be internalized through a process

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such as macropinoctyosis. Collectively, these results indicate the existence of at least two

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mechanisms (clathrin-mediated endocytosis and macropinocytosis) by which premigratory

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cranial neural crest cells reduce levels of surface Cad6B.

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To test these hypothesized mechanisms, we pharmacologically inhibited endocytosis and

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macropinoctyosis in neural crest cell explants. We first assessed a role for dynamin by treating

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explants with Dynasore, a dynamin inhibitor (Macia et al., 2006). Our results show that,

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compared to control explants (Fig. 8A-A”, arrowhead, arrow indicates Cad6B membrane

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staining), dynamin inhibition blocks neural crest cell EMT and migration (23/24 explants), as


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evidenced by brightfield images and phalloidin staining (arrows in Fig. S2, see Fig S2B, B’;

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Coles et al., 2007; Taneyhill et al., 2007; Jhingory et al., 2010) compared to control explants that

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underwent EMT/migrated normally (Fig. S2A, A’, 18/21 explants examined). Moreover,

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Dynasore treatment leads to a statistically significant 2.2-fold increase in the accumulation of

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multiple, large intracellular and membrane-bound Cad6B puncta greater than or equal to 2µm

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compared to control explants (Fig. 8B-B”, arrowheads; Fig. S2G). To determine a function for

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macropinocytosis in internalizing Cad6B during neural crest cell EMT, we prevented

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macropinosome formation by blocking actin polymerization (Mercer and Helenius, 2009) using

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Latrunculin A (Spector et al., 1983; Mercer and Helenius, 2009). Latrunculin A treatment of

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explants eliminated actin polymerization as evidenced by the absence of phalloidin staining (Fig.

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8C’) compared to control explants (Fig. S2A’). Importantly, addition of Latrunculin A severely

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decreased the presence/number of Cad6B puncta (Fig. 8C-C”, Fig. S2G), with the majority of

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Cad6B still localized to the plasma membrane (Fig. 8C”, arrows), in contrast to Cad6B

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distribution observed in control explants (Fig. 8A-A”, arrows, arrowheads). Furthermore,

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Latrunculin A treatment inhibited EMT (Fig. S2C, C’, 12/12 explants examined) compared to

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normal EMT/migration in control explants. We next tested the sensitivity of Cad6B

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internalization to the amiloride EIPA, which is a macropinocytosis inhibitor (Mercer and

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Helenius, 2008; Koivusalo et al., 2010). EIPA treatment resulted in the appearance of multiple,

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large Cad6B cytoplasmic puncta (2.5-fold increase in the number of large Cad6B puncta; Fig.

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S2G) co-localizing with p120-catenin (Fig. 8D-D”, arrowheads) and abolished EMT (Fig. S2D,

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D’; 18/23 explants show an absence of EMT/migration compared to normal EMT/migration in

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18/21 control explants). Finally, the size of the accumulated puncta (1.5-3μm, ~700 puncta

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measured) is also indicative of macropinocytosis (Swanson, 1989; Hewlett et al., 1994;

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Schnatwinkel et al., 2004).

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To rule out potential non-specific effects of Dynasore and EIPA on Cad6B and EMT, we

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used two additional chemicals that block targets of Dynasore and EIPA: dansylcadaverine, a

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clathrin-specific inhibitor (for Dynasore) (Davies et al., 1980; Bradley et al., 1993; Wang and

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Liu, 2003), and NSC23766, a Rac1-specific inhibitor (for EIPA) (Gao et al., 2004). Indeed,

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addition of these inhibitors partially recapitulated the phenotype observed upon treatment of

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explants with either EIPA or Dynasore, including a reduction in EMT in the case of NSC23766

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(14/14 explants; compare Fig. S2D-D’ and F-F’), whereas the majority of explants (12/15)

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addition, treatment with dansylcadaverine or NSC23766 caused accumulation of Cad6B puncta

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(Fig. S3A-A’, B-B’, arrows), although not statistically significant (Fig. S2G). These data

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strongly suggest that Cad6B undergoes macropinocytosis and clathrin-dependent endocytosis in

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premigratory neural crest cells.

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We then examined the distribution of p120-catenin with respect to Cad6B upon inhibitor

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treatment. Surprisingly, many of the Cad6B cytoplasmic puncta observed upon Dynasore (Fig.

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8B”, carets) or EIPA (Fig. 8D”, carets) treatment were p120-catenin-negative, in contrast to our

Accepted manuscript

underwent normal EMT in the presence of dansylcadaverine (compare Fig. S2B-B’ and E-E’). In

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expectation that these would possess both Cad6B and p120-catenin. To investigate if these

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observations were due to extraneous inhibitor effects, we examined Cad6B and p120-catenin co-

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localization in untreated cells, explants, and embryos. We noticed in several instances that

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Cad6B did not co-localize with p120-catenin in puncta in untreated cells (Fig. 3B”, D”, carets),

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explants (Fig. 8A”, carets), and embryos (Fig. 7B”, carets), implying that these are either

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endocytic vesicles or that p120-catenin dissociates later from the puncta post-macropinocytosis.

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Taken together, our results reveal that Cad6B is internalized during cranial neural crest cell EMT


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through dynamin-dependent, clathrin-mediated endocytosis and macropinocytosis, with the

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absence of internalization severely impacting the ability of cranial neural crest cells to undergo

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EMT and migrate.

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DISCUSSION

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Regulation of cadherin proteins is critical during development and disease (Lim and Thiery,

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2012; Gheldof A, 2013). In this study, we provide the first evidence for dynamin-dependent

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cadherin endocytosis and macropinocytosis during an in vivo EMT. By following the processing

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of endogenous Cad6B protein using an antibody, we demonstrate that Cad6B undergoes

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internalization in premigratory and early migratory neural crest cells. Interestingly, we note no

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differences in the degree of endocytosis during specific stages of EMT, and this internalization

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process may be conserved, as we observe internalized Cad6B in both chick hindbrain and trunk

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premigratory neural crest cells (Fig. S4). We further show that Cad6B possesses a functional

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motif in its cytoplasmic domain that negatively regulates Cad6B endocytosis, likely due to the

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presence of bound p120-catenin. Partial co-localization of internalized Cad6B with clathrin


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suggests that Cad6B undergoes endocytosis through a clathrin-mediated pathway, and retention

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of Cad6B on the plasma membrane in large puncta/vesicles upon addition of a dynamin inhibitor

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further substantiates this observation. Interestingly, many intracellular puncta are in fact devoid

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of clathrin but instead double-positive for Cad6B and p120-catenin, suggesting that whole

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adherens junctions are internalized through an additional mechanism such as macropinocytosis.

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A reduction in Cad6B puncta in neural crest cells treated with Latrunculin A confirms this

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hypothesis. In addition, results from EIPA treatment lend further credence to a role for

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macropinocytosis in Cad6B internalization, a process that also depends upon dynamin.

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Importantly, treatment with either Dynasore, Latrunculin A, or EIPA blocked neural crest cell

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EMT and migration, suggesting that down-regulation of Cad6B through endocytosis and

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macropinocytosis is critical for EMT. Nevertheless, the use of such chemical inhibitors could

340

have broad-spectrum effects on cell physiology and function (reviewed in (Ivanov, 2008)) and

341

impact Cad6B distribution and/or EMT. For example, EIPA can alter morphology and the

342

intracellular distribution of early and late endosomes in HeLa cells (Fretz et al., 2006). Dynasore,

343

which affects clathrin-mediated endocytosis, can also have minor effects on caveolar endocytic

344

pathways (Macia et al., 2006). To rule out any non-specific effects with regards to these

345

compounds, Cad6B localization was investigated in the presence of more specific inhibitors

346

(Dansylcadaverine for clathrin, NSC23766 for Rac1). The use of these inhibitors partially

347

recapitulated the Cad6B accumulation phenotype noted in the presence of Dynasore and EIPA,

348

although the effects on EMT varied. Thus, the accumulation of large Cad6B puncta, even under

349

these more specific inhibitor conditions, validates the phenotypes observed after EIPA and

350

Dynasore treatment. Lastly, these internalization processes appear to be specific to Cad6B, as no

351

effects on N-cadherin were observed due to the absence of N-cadherin in cranial neural crest

352

cells during EMT (Dady et al., 2012; Rogers et al., 2013). Therefore, a combination of

353

endocytosis and macropinocytosis, together with proteolysis (Schiffmacher et al., 2014), may be

354

required to fully remove Cad6B from the plasma membrane of premigratory cranial neural crest

355

cells undergoing EMT.

356

The mechanism(s) by which Cad6B is removed from premigratory neural crest cell

357

membranes has broad implications for cell biology, tissue homeostasis, and pathogenic

358

conditions resulting from de-regulated cadherins (Gheldof A, 2013). We have previously shown

359

that one mechanism by which Cad6B is lost from premigratory cranial neural crest cells is

361

to cytoplasmic puncta in premigratory and early migratory cranial neural crest cells, suggestive

362

of a potential role for Cad6B internalization as neural crest cells undergo EMT. The possibility

363

that internalization of Cad6B plays an important role during EMT is not without precedence, as

364

cadherins undergo endocytosis during a variety of other cell biological processes (Cavey and

365

Lecuit, 2009; Kowalczyk and Nanes, 2012; Collinet and Lecuit, 2013), but cadherin

366

internalization during EMT has yet to be reported until now.

367

Cadherins possess endocytosis motifs in their cytoplasmic domain that regulate their

Accepted manuscript

proteolytic cleavage (Schiffmacher et al., 2014). Nevertheless, we still observed Cad6B localized

368

plasma membrane-bound state (Xiao et al., 2005; Miyashita and Ozawa, 2007a; Miyashita and

369

Ozawa, 2007b; Tai et al., 2007; Hong et al., 2010; Ishiyama et al., 2010; Kowalczyk and Nanes,

370

2012; Nanes et al., 2012). Our experiments reveal that Cad6B possesses a putative p120-catenin

371

binding motif in its cytoplasmic domain that functions to negatively regulate Cad6B endocytosis.

372

p120-catenin modulates cadherin endocytosis by “masking” the dileucine motif (Miyashita and

373

Ozawa, 2007b; Ishiyama et al., 2010; Nanes et al., 2012). Dissociation of p120-catenin from the

374

cadherin exposes the dileucine motif, which in turn is recognized by cytoplasmic adaptor


360

375

proteins of the clathrin-mediated endocytic pathway and leads to cadherin endocytosis

376

(Miyashita and Ozawa, 2007b; Ishiyama et al., 2010; Nanes et al., 2012). Mutating this putative

377

p120-catenin binding motif to alanine enhanced endocytosis of Cad6B, in agreement with results

378

for E-cadherin (Xiao et al., 2003a; Miyashita and Ozawa, 2007b). Furthermore, this mutant was

379

deficient in forming stable cell-cell adhesions and possessed reduced overall stability. Enhanced

380

endocytosis could translate into fewer opportunities for this mutant Cad6B to form stable

381

interactions with the cytoskeleton, further augmenting its endocytosis and decreasing its stability,

382

as observed for other cadherins in vitro (Collinet and Lecuit, 2013). Alternatively, association

383

with p120-catenin could be required for cadherins to interact with the cytoskeleton (Hoshino et

384

al., 2005), which may lead to similar effects upon the absence of p120-catenin binding.

385

The dileucine motif in Cad6B, which is actually an LI motif and corresponds to the

386

consensus (DE)XXXL(LI) or the DXXLL, is also critical for endocytosis (Bonifacino and Traub,

387

2003). This motif (normally masked by p120-catenin) binds several proteins required for

388

clathrin-mediated endocytosis (Ishiyama et al., 2010), and thus the absence of this motif was

389

hypothesized to negatively affect Cad6B endocytosis. Mutating the LI residues in Cad6B,


390

however, did not significantly affect Cad6B internalization and its association with the

391

cytoskeleton. This is in contrast with what has been observed with E-cadherin (Miyashita and

392

Ozawa, 2007a) and N-cadherin (Tai et al., 2007), and could be due to the lack of acidic residues

393

that usually precede this dileucine motif in these other cadherins (Bonifacino and Traub, 2003).

394

Furthermore, the lysine at position 748 in E-cadherin controls E-cadherin endocytosis in vitro,

395

and complete loss of endocytosis was only observed when both the dileucine motif and lysine

396

residue were mutated (Hong et al., 2010). Because Cad6B has a conserved lysine in a similar

397

position, mutating it along with the LI motif may be necessary to block Cad6B endocytosis, and

398

thus co-localization with clathrin should serve as indirect evidence for the role of the LI motif

399

and lysine during Cad6B endocytosis. Nonetheless, many intracellular puncta observed in vivo

400

and in vitro were not double-positive for clathrin and Cad6B, implying additional mechanisms of

401

Cad6B internalization.

402

Our results now reveal that, besides clathrin-mediated endocytosis, Cad6B undergoes

403

internalization as part of whole adherens junctions through macropinocytosis. The use of

404

macropinocytosis to remove cadherins from membranes has been shown previously in vitro for

405

E-cadherin molecules not actively engaged in cell-cell adhesion (Bryant et al., 2007) and for N-

406

cadherin at the leading edge of migratory cells (Sharma and Henderson, 2007), but to our

407

knowledge has never been documented during an in vivo EMT. As such, our work is the first to

408

report the significance of cadherin internalization during neural crest cell EMT. Importantly, this

409

study raises additional questions regarding the fate of internalized cadherins and catenins during

410

neural crest cell EMT. For example, the absence of p120-catenin in some intracellular puncta

411

may be indicative of the need to release p120-catenin so that it can perform other signaling roles

412

that impinge upon neural crest cell EMT (Bellovin et al., 2005; Yanagisawa and Anastasiadis,

413

2006; Cheung et al., 2010; Johnson et al., 2010). Furthermore, it is intriguing that cranial neural

414

crest cells employ multiple mechanisms (internalization and proteolysis) to reduce surface

415

Cad6B levels during EMT. One possibility is that these mechanisms are used in different sub-

416

populations of cranial neural crest cells (Lee et al., 2013; Ridenour et al., 2014). Alternatively,

417

this rapid reduction in Cad6B protein may be necessary to release catenins so they can signal

418

(i.e., transcription) and/or form complexes with other mesenchymal cadherins to mediate

419

migration. Finally, en masse removal of Cad6B from premigratory cranial neural crest cells


420

could be occurring simply to eliminate physical barriers (e.g., adherens junctions) that initially

421

hold premigratory neural crest cells together prior to their delamination and EMT.

422

In summary, our results provide crucial insight into additional molecular mechanisms

423

underlying the post-translational down-regulation of cadherins in premigratory cranial neural

424

crest cells. Our studies are the first to delineate the importance of Cad6B internalization during

425

an in vivo EMT in the neural crest. Notably, this internalization of Cad6B occurs through at least

426

two mechanisms, clathrin-mediated endocytosis and macropinocytosis, both of which are

427

dynamin-dependent processes. Furthermore, Cad6B internalization is critical for cranial neural

428

crest cells to undergo EMT and properly migrate. Collectively, our work highlights how cranial

429

neural crest cells employ multiple means to effectively clear this important cadherin from their

430

plasma membranes and initiate EMT in the developing vertebrate embryo.

431 432

MATERIALS AND METHODS

433

Chick embryos

434

Fertilized chicken eggs were obtained from B & E Farms (York, PA, USA) and incubated at

435

38°C in humidified incubators (EggCartons.com, Manchaug, MA, USA). Embryos were staged

436

by the number of pairs of somites (somite stage, ss) according to Hamburger-Hamilton

437

(Hamburger and Hamilton, 1992).

438

Neural crest cell explant assays

439

Neural crest cell explants were prepared as described in (Coles et al., 2007; Taneyhill et al.,

440

2007; Jhingory et al., 2010). Briefly, dorsal neural folds (containing premigratory neural crest

441

cells) from chick embryo midbrains were dissected out into PB-1 standard medium and placed

442

into chamber slides coated with a 1:100 dilution of poly-L-lysine (P5899, Sigma, St. Louis, MO,

443

USA) and fibronectin (356008, Corning, NY, USA). Cultures were incubated in serum-free

444

Dulbecco’s Modified Eagle’s Medium (DMEM, 10-013-CV, CellGro, Manassas, VA, USA)

445

supplemented with a 1:100 dilution of N-2 (17502-048, Life Technologies, Carlsbad, CA, USA)

446

at 37˚C for varying times. For inhibitor assays, tissue was directly explanted into chamber slides

447

containing 0.5% (vol/vol) DMSO, Dynasore (100µM; 304448-55-3, Adipogen, San Diego, CA,


448

USA), Latrunculin A (300nM; L5163, Sigma-Aldrich, St. Louis, MO, USA), EIPA (50µM;

449

3378, Tocris Bioscience, Sunnyvale, CA, USA), Dansylcadaverine (100µM; sc-214851, SCBT,

450

Santa Cruz, USA), and NSC23766 (150µM; sc-204823, SCBT, Santa Cruz, USA). Explants

451

were incubated for 3.5 hours at 37˚C to allow for neural crest cells to undergo EMT, followed by

452

fixation and immunostaining (described below). Putative macropinocytic vesicles in explants

453

were visualized in 3D using the Zen software (Zeiss), with vesicle length measured at its greatest

454

extent using the software Measurement tool.

455

Cloning of Cad6B mutants

456

Full-length Cad6B cloned in pCIG (Coles et al., 2007) was sub-cloned into pCI-H2B-RFP (gift

457

from Dr. M. Bronner) along with a hemagglutinin (HA) epitope tag at its C-terminus through

458

standard cloning procedures. To create endocytic mutants, Cad6B in pCI-H2B-RFP was

459

mutagenized using the QuikChange II XL Site-Directed Mutagenesis Kit (200521, Agilent

460

Technologies, Santa Clara, CA, USA). All constructs were sequenced to ensure sequence

461

accuracy.

462

FlpIn cell culture and reagents

463

CHO cells stably expressing a single integrated copy of wild-type and various endocytic mutants

464

of Cad6B were created as described previously using the FlpIn system (K6010-02, R75807, Life

465

Technologies, Carlsbad, CA, USA; (Schiffmacher et al., 2014)). Briefly, HA-tagged wild-type or

466

mutant Cad6B was directionally sub-cloned from pCI-H2B-RFP (above) into the pcDNA5/FRT

467

vector and co-transfected with pOG44 into FlpIn-CHO cells. After transfection, cells were

468

trypsinized and plated at 20–25% confluency in F12 media supplemented with 1 mM L-

469

glutamine and 600μg/ml hygromycin for selection of positive transfectants (cells were

470

subsequently grown in this medium). Individual colonies were transferred to 96 wells and

471

sequentially passaged into 48-, 24-, and 12-plates. Colonies were screened for Cad6B expression

472

by immunohistochemistry, and those with maximum transfection efficiency were eventually

473

passaged to 10-cm plates and expression verified with immunoblotting. Cells were grown at

474

37˚C in Ham’s F12 growth media supplemented with 10% fetal bovine serum (S11150, Atlanta

475

Biologicals, Flowery Branch, GA, USA), 600μg/mL hygromycin (30-240-R, CellGro, Manassas,

476

VA, USA), 1mM L-glutamine (25-005, CellGro, Manassas, VA, USA), and 1:100 dilution of 1:1

477

penicillin-streptomycin (30-002-CI, CellGro, Manassas, VA, USA).

478


Accepted manuscript

479 480

Immunohistochemistry

481

6 and 7ss embryos were collected and fixed in 4% paraformaldehyde (PFA) for 30 minutes at

482

room temperature. Embryos were permeabilized in fresh Tris-buffered saline (TBS) containing

483

0.2% TX-100 (TBST) and blocked in this plus 5% fetal bovine serum (FBS) for one hour at

484

room temperature. Immunostaining was performed overnight at 4˚C with Cad6B primary and

485

anti-mouse Cad6B secondary antibodies as in (Jhingory et al., 2010; Schiffmacher et al., 2014).

486

Explants were fixed by serial dilution of the media with 4% PFA for 20 minutes at room

487

temperature, permeabilized with fresh TBST, blocked with 5% FBS in TBST, and incubated

488

with the Cad6B primary antibody overnight at 4˚C and secondary antibody for three hours at

489

room temperature. Cells were fixed in either 4% PFA for 20 minutes at room temperature or in

490

cold 5% ethanol/95% acetic acid for 20 minutes at -20˚C. For cells fixed in 4% PFA,

491

permeabilization was carried out with TBST, and immunostaining was performed as for

492

explants. Immunostaining on cells fixed in ethanol/acetic acid was performed similarly as for

493

explants but in TBS buffer without detergent.

494

The following primary antibodies and concentrations were used in experiments (fixation

495

conditions provided in the next section): Cad6B (1:100 in whole-mount and 1:250 on sections

496

and cells: Cad6B (CCD6B-1, Developmental Studies Hybridoma Bank, OH, USA; referred to as

497

NT-6B in the text), caveolin-1 (1:500; ab2910, Abcam, Cambridge, England), HA (1:750; 3F10,

498

Roche, Basel, Switzerland), clathrin (1:750; ab21679, Abcam, Cambridge, England),

499

Na+K+ATPase (1:750; ab76020, Abcam, Cambridge, UK), p120-catenin (1:500; sc-373751;

500

Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (1:500; AHO0462, Life

501

Technologies, Carlsbad, CA, USA), GFP (1:1000; R10367, Life Technologies, Carlsbad, CA,

502

USA), and K-cad (1:200; ab64917, Abcam, Cambridge, UK; referred to as CT-6B in the text).

503

Alexa Fluor488-conjugated phalloidin (Life Technologies, Carlsbad, CA, USA) was used at

504

1:50. Appropriate fluorescently-conjugated secondary antibodies (AlexaFluor 488, 594, 647;

505

Life Technologies, Carlsbad, CA, USA) were used at the following concentrations for each

506

primary antibody: Cad6B (1:250 in whole-mount and 1:500 on cells), HA (1:1000), clathrin

507

(1:1000), Na+K+ATPase (1:1000), p120 (1:1000), β-catenin (1:1000), GFP (1:1500), and K-cad

508

(1:750).


Accepted manuscript

509 510

Cad6B antibody feeding assays

511

The feeding assay was performed in vivo as in (Arancibia-Carcamo IL, 2006) with the following

512

modifications. The neural tube lumen of 5ss embryos was filled with CCD6B-1 antibody after

513

electroporation of a membrane-GFP construct to label plasma membranes (a kind gift from Dr.

514

Paul Kulesa). After incubation to allow for EMT, the embryos were fixed and immunostaining

515

was performed for Cad6B and GFP as described above.

516

For the in vitro feeding assay, FlpIn-wtCad6B cells were washed 3X with ice-cold fresh

517

PBS2+ (PBS supplemented with 1.5mM MgCl2 and 0.2mM CaCl2) and incubated with a 1:50

518

dilution of NT-6B antibody in media at 4˚C for an hour. The cells were washed 3X with ice-cold

519

PBS2+, replaced with warm media, and incubated at 37˚C for an hour. Following this, cells were

520

brought back to 4˚C and subjected to 3X acid wash (0.1M glycine, pH 2.0) for 5 minutes at 4˚C.

521

The cells were washed 2X with PBS, fixed, and immunostaining was performed as described

522

above.

523

To perform the feeding assay ex vivo, cranial dorsal neural folds from 5ss embryos were

524

explanted as described above and incubated at 37˚C for 90 minutes to allow tissue to attach to

525

the chamber slide. The media was serially diluted with ice-cold fresh PBS2+, and explants were

526

incubated at 4˚C for 5 minutes. PBS2+ was then serially diluted with ice-cold explant media

527

containing a 1:50 dilution of CCD6B-1 antibody and incubated at 4˚C for an hour. Following

528

serial dilution with ice-cold PBS2+, fresh warm explant media was added to the explants, which

529

were then incubated at 37˚C for three hours. The explants were brought back to 4˚C and,

530

following serial dilution of the explant media with ice-cold PBS2+, non-internalized antibody

531

(bound to the plasma membrane) was removed by treatment with the low pH buffer described

532

above for 15 minutes at 4˚C temperature. The explants were fixed, permeabilized, and

533

immunostaining was performed as described above.

534

Protein extraction and immunoblotting


535

Protein extraction and immunoblotting was performed as described in (Schiffmacher et al.,

536

2014). Briefly, cells were scraped in ice-cold PBS2+ and pelleted with low-speed centrifugation.

537

Cell pellets were lysed in 2X volume of lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 1%

538

IGEPAL CA-630) supplemented with protease inhibitors for 30 minutes at 4˚C with periodic

539

mixing. Following separation of supernatant from insoluble material through high-speed

540

centrifugation, supernatant protein concentration was quantified by a Bradford assay. Equivalent

541

amounts of protein per sample were boiled at 95°C for 5 minutes in 4X reducing Laemmli

542

sample buffer, processed by SDS–PAGE, and then transferred to PVDF membrane. Membranes

543

were blocked in 5% non-fat milk in PBS supplemented with 0.1% Tween and incubated

544

overnight at 4°C with the following primary antibodies diluted in blocking solution: Cad6B

545

(1:80, CCD6B-1), β-actin (1:1000; sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA),

546

and HA (1:1000; 3F10, Roche, Basel, Switzerland). Membranes were washed and incubated with

547

species- and isotype-specific horseradish peroxidase-conjugated secondary antibodies (40ng/ml;

548

Jackson ImmunoResearch, West Grove, PA, USA) in blocking solution for 1 hour at room

549

temperature. Antibody detection was performed using Supersignal West Pico or Femto

550

chemiluminescent substrate (Thermo Pierce Scientific, Waltham, MA, USA) following washes

551

and visualized using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). Band volumes

552

(intensities) were calculated from unmodified immunoblot images using Image Lab software

553

(Bio-Rad, Hercules, CA, USA), and analyzed by repeated measures analysis of variance

554

assuming a compound symmetric covariance matrix for time and an unstructured covariance

555

matrix for band intensity based on AIC values within the PROC MIXED procedure (SAS

556

statistical software, SAS Institute, Cary, NC). Levels were deemed significantly different when p

557

< 0.05 based on Fisher’s LSD multiple mean comparison test. Confidence intervals were

558

calculated for the cytoskeleton (CSK) extraction assays and were deemed statistically significant

559

if they did not overlap.

560

Cell surface biotinylation

561

Confluent layers of cells were brought to 4˚C and washed twice with ice-cold PBS2+. After

562

removal of PBS2+, EZ-Link Sulfo-NHS-Biotin (21326; Thermo Scientific, Waltham, MA, USA)

563

dissolved in ice-cold PBS2+ was added to cells at 1mg/mL, and cells were incubated on a rocking

564

platform for 30 minutes at 4˚C. Excess biotin was quenched by washing 3X, 5 minutes each,


565

with ice-cold PBS2+ supplemented with 100mM glycine and 0.5% BSA. After rinsing off

566

residual quenching buffer, cells were scraped off the dish in ice-cold PBS2+, pelleted at 2300rpm

567

for 5 minutes at 4˚C, and set aside as positive control (“Total Surface” fraction). Biotin was then

568

stripped off of cells from another plate by washing cells 3X, 10 minutes each, at 4˚C using a

569

stripping buffer (75mM NaCl, 75mM NaOH, 50mM L-glutathione (0399, Amresco, Solon, OH,

570

USA) supplemented with 1% BSA), followed by cell scraping in ice-cold PBS2+, and the cell

571

pellets set aside (negative control). Warm growth media (see above) was added to cells in the

572

remaining two plates, and cells were incubated at 37˚C for 60 and 180 minutes. At the indicated

573

time points, cells were brought back to 4˚C, and any remaining biotin from cell surface-bound,

574

non-endocytosed proteins was removed with the biotin stripping conditions described above.

575

Cells were lysed and subjected to a Bradford protein analysis as described previously.

576

Streptavidin-Agarose resin (20347, Thermo Scientific, Waltham, MA, USA) pre-blocked with

577

4% BSA was incubated with 175-200μg of total protein lysate overnight at 4˚C to

578

immunoprecipitate biotin-labeled proteins. After washing 3X with 1X lysis buffer, 4X Laemmli

579

sample buffer was added to the resin, and samples were boiled at 95˚C for 5 minutes and loaded

580

onto a 7.5% PAGE gel. Electrophoresis, immunoblotting, and band intensity quantification for

581

Cad6B was performed as described above.

582

TX-100 extraction of soluble and insoluble proteins

583

Ice-cold cytoskeleton extraction buffer (CSK) (50mM NaCl, 10mM Pipes pH 6.8, 3mM MgCI2,

584

0.5%Triton X-100, 300mM sucrose), supplemented with 1.2mM PMSF (36978; Thermo Pierce,

585

Waltham, MA, USA) and 1X dilution of cOmplete Protease Inhibitor Cocktail (04693124001;

586

Roche, Basel, Switzerland), was added to equivalent numbers of cells and incubated on a rocking

587

platform for 30 minutes at 4˚C. The extraction buffer was collected, centrifuged at maximum

588

speed for 5 minutes at 4˚C, and the supernatant designated the TX-100 soluble fraction. The cells

589

remaining on the plate were scraped and pelleted in ice-cold PBS2+, and 2X Laemmli sample

590

buffer was directly added to the cell pellet. Following brief pulses of sonication to shear genomic

591

DNA, the lysate was centrifuged at maximum speed for 5 minutes at 4˚C, and the supernatant

592

designated the TX-100 insoluble fraction. Both fractions were boiled at 95°C for 5 minutes and

593

subjected to electrophoresis, immunoblotting, and band intensity quantification as described

594

previously.

595

Confocal microscopy

596

All images were acquired with the LSM Zeiss 710 microscope (Carl Zeiss Microscopy,

597

Thornwood, NY, USA) at the University of Maryland Imaging Core Facility. Where possible,

598

the laser power, gain, and offset were kept consistent for different channels, and the pinhole was

599

always set to one airy unit. The Z-section optical images were acquired between 0.25-0.4μm per

600

optical section and reconstituted in 3D composites using the Zen software (Zeiss).


Accepted manuscript

601 602

ACKNOWLEDGEMENTS

603

We thank Dr. Paul Kulesa, Stowers Institute of Medical Research, for the membrane GFP

604

construct; Dr. Ashley Franklin, Point Defiance Zoo and Aquarium, for assistance with statistics;

605

Dr. Shruthi Rangarajan for help with figures; and Ms. Lizbeth Hu for technical assistance. This

606

work was supported by NIH grant R00HD055034 to L.A.T. and a Sigma Xi Grants-In-Aid of

607

Research and the University of Maryland Ann G. Wylie Dissertation Fellowship to R. P.

608 609

REFERENCES

610

Akhtar, N. and Hotchin, N. A. (2001). RAC1 regulates adherens junctions through endocytosis

611

of E-cadherin. Molecular biology of the cell 12, 847-862.

612

Arancibia-Carcamo IL, F. B., Moss SJ, Kittler JT. (2006). Studying the Localization, Surface

613

Stability and Endocytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation

614

Approaches. In The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology (ed.

615

K. J. a. M. SJ), pp. 91: Taylor & Francis Group, LLC.

616

Bellovin, D. I., Bates, R. C., Muzikansky, A., Rimm, D. L. and Mercurio, A. M. (2005).

617

Altered localization of p120 catenin during epithelial to mesenchymal transition of colon

618

carcinoma is prognostic for aggressive disease. Cancer research 65, 10938-10945.

619

Bonifacino, J. S. and Traub, L. M. (2003). Signals for sorting of transmembrane proteins to

620

endosomes and lysosomes. Annual review of biochemistry 72, 395-447.

621

Bradley, J. R., Johnson, D. R. and Pober, J. S. (1993). Four different classes of inhibitors of

622

receptor-mediated endocytosis decrease tumor necrosis factor-induced gene expression in human

623

endothelial cells. Journal of immunology 150, 5544-5555.


624

Bryant, D. M., Wylie, F. G. and Stow, J. L. (2005). Regulation of endocytosis, nuclear

625

translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin. Molecular

626

biology of the cell 16, 14-23.

627

Bryant, D. M., Kerr, M. C., Hammond, L. A., Joseph, S. R., Mostov, K. E., Teasdale, R. D.

628

and Stow, J. L. (2007). EGF induces macropinocytosis and SNX1-modulated recycling of E-

629

cadherin. Journal of cell science 120, 1818-1828.

630

Cain, C. C., Sipe, D. M. and Murphy, R. F. (1989). Regulation of endocytic pH by the

631

Na+,K+-ATPase in living cells. Proceedings of the National Academy of Sciences of the United

632

States of America 86, 544-548.

633

Cao, H., Chen, J., Awoniyi, M., Henley, J. R. and McNiven, M. A. (2007). Dynamin 2

634

mediates fluid-phase micropinocytosis in epithelial cells. Journal of cell science 120, 4167-4177.

635

Cavey, M. and Lecuit, T. (2009). Molecular bases of cell-cell junctions stability and dynamics.

636

Cold Spring Harbor perspectives in biology 1, a002998.

637

Cheung, L. W., Leung, P. C. and Wong, A. S. (2010). Cadherin switching and activation of

638

p120 catenin signaling are mediators of gonadotropin-releasing hormone to promote tumor cell

639

migration and invasion in ovarian cancer. Oncogene 29, 2427-2440.

640

Chiasson, C. M., Wittich, K. B., Vincent, P. A., Faundez, V. and Kowalczyk, A. P. (2009).

641

p120-catenin inhibits VE-cadherin internalization through a Rho-independent mechanism.

642

Molecular biology of the cell 20, 1970-1980.

643

Classen, A. K., Anderson, K. I., Marois, E. and Eaton, S. (2005). Hexagonal packing of

644

Drosophila wing epithelial cells by the planar cell polarity pathway. Developmental cell 9, 805-

645

817.

646

Coles, E. G., Taneyhill, L. A. and Bronner-Fraser, M. (2007). A critical role for Cadherin6B

647

in regulating avian neural crest emigration. Developmental biology 312, 533-544.

648

Collinet, C. and Lecuit, T. (2013). Stability and dynamics of cell-cell junctions. Progress in

649

molecular biology and translational science 116, 25-47.

650

Dady, A., Blavet, C. and Duband, J. L. (2012). Timing and kinetics of E- to N-cadherin switch

651

during neurulation in the avian embryo. Developmental dynamics : an official publication of the

652

American Association of Anatomists 241, 1333-1349.


653

Davies, P. J., Davies, D. R., Levitzki, A., Maxfield, F. R., Milhaud, P., Willingham, M. C.

654

and Pastan, I. H. (1980). Transglutaminase is essential in receptor-mediated endocytosis of

655

alpha 2-macroglobulin and polypeptide hormones. Nature 283, 162-167.

656

Davis, M. A., Ireton, R. C. and Reynolds, A. B. (2003). A core function for p120-catenin in

657

cadherin turnover. The Journal of cell biology 163, 525-534.

658

Desclozeaux, M., Venturato, J., Wylie, F. G., Kay, J. G., Joseph, S. R., Le, H. T. and Stow,

659

J. L. (2008). Active Rab11 and functional recycling endosome are required for E-cadherin

660

trafficking and lumen formation during epithelial morphogenesis. American journal of

661

physiology. Cell physiology 295, C545-556.

662

Duband, J. L., Volberg, T., Sabanay, I., Thiery, J. P. and Geiger, B. (1988). Spatial and

663

temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during

664

avian embryogenesis. Development 103, 325-344.

665

Dupin, N. L. D. a. E. (2014). The Neural Crest, a Fourth Germ Layer of the Vertebrate Embryo:

666

Significance in Chordate Evolution. In NEURAL CREST CELLS: Evolution, Development and

667

Disease (ed. P. Trainor), pp. 4-22: Elsevier.

668

Farquhar, M. G. and Palade, G. E. (1963). Junctional complexes in various epithelia. The

669

Journal of cell biology 17, 375-412.

670

Feldmann, T., Glukmann, V., Medvenev, E., Shpolansky, U., Galili, D., Lichtstein, D. and

671

Rosen, H. (2007). Role of endosomal Na+-K+-ATPase and cardiac steroids in the regulation of

672

endocytosis. American journal of physiology. Cell physiology 293, C885-896.

673

Fishwick, K. J., Neiderer, T. E., Jhingory, S., Bronner, M. E. and Taneyhill, L. A. (2012).

674

The tight junction protein claudin-1 influences cranial neural crest cell emigration. Mechanisms

675

of development.

676

Fretz, M., Jin, J., Conibere, R., Penning, N. A., Al-Taei, S., Storm, G., Futaki, S., Takeuchi,

677

T., Nakase, I. and Jones, A. T. (2006). Effects of Na+/H+ exchanger inhibitors on subcellular

678

localisation of endocytic organelles and intracellular dynamics of protein transduction domains

679

HIV-TAT peptide and octaarginine. Journal of controlled release : official journal of the

680

Controlled Release Society 116, 247-254.

681

Fuchs, R., Schmid, S. and Mellman, I. (1989). A possible role for Na+,K+-ATPase in

682

regulating ATP-dependent endosome acidification. Proceedings of the National Academy of

683

Sciences of the United States of America 86, 539-543.


684

Gao, Y., Dickerson, J. B., Guo, F., Zheng, J. and Zheng, Y. (2004). Rational design and

685

characterization of a Rac GTPase-specific small molecule inhibitor. Proceedings of the National

686

Academy of Sciences of the United States of America 101, 7618-7623.

687

Gheldof A, B. G. (2013). Cadherins and Epithelial-to-Mesenchymal Transition: Elsevier.

688

Hall. (2009). The Neural Crest and Neural Crest Cells in Vertebrate Development and

689

Evolution: Springer.

690

Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the development of

691

the chick embryo. 1951. Developmental dynamics : an official publication of the American

692

Association of Anatomists 195, 231-272.

693

Hatta, K. and Takeichi, M. (1986). Expression of N-cadherin adhesion molecules associated

694

with early morphogenetic events in chick development. Nature 320, 447-449.

695

Hay, E. D. (1995). An overview of epithelio-mesenchymal transformation. Acta anatomica 154,

696

8-20.

697

Hewlett, L. J., Prescott, A. R. and Watts, C. (1994). The coated pit and macropinocytic

698

pathways serve distinct endosome populations. The Journal of cell biology 124, 689-703.

699

Hong, S., Troyanovsky, R. B. and Troyanovsky, S. M. (2010). Spontaneous assembly and

700

active disassembly balance adherens junction homeostasis. Proceedings of the National Academy

701

of Sciences of the United States of America 107, 3528-3533.

702

Hoshino, T., Sakisaka, T., Baba, T., Yamada, T., Kimura, T. and Takai, Y. (2005).

703

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn. The Journal

704

of biological chemistry 280, 24095-24103.

705

Ireton, R. C., Davis, M. A., van Hengel, J., Mariner, D. J., Barnes, K., Thoreson, M. A.,

706

Anastasiadis, P. Z., Matrisian, L., Bundy, L. M., Sealy, L. et al. (2002). A novel role for p120

707

catenin in E-cadherin function. The Journal of cell biology 159, 465-476.

708

Ishiyama, N., Lee, S. H., Liu, S., Li, G. Y., Smith, M. J., Reichardt, L. F. and Ikura, M.

709

(2010). Dynamic and static interactions between p120 catenin and E-cadherin regulate the

710

stability of cell-cell adhesion. Cell 141, 117-128.

711

Ivanov. (2008). Pharmacological Inhibition of Endocytic Pathways: Is It Specific Enough to Be

712

Useful? In Exocytosis and Endocytosis (ed. I. AI), pp. 15-33: Humana Press.


713

Jarrett, O., Stow, J. L., Yap, A. S. and Key, B. (2002). Dynamin-dependent endocytosis is

714

necessary for convergent-extension movements in Xenopus animal cap explants. The

715

International journal of developmental biology 46, 467-473.

716

Jhingory, S., Wu, C. Y. and Taneyhill, L. A. (2010). Novel insight into the function and

717

regulation of alphaN-catenin by Snail2 during chick neural crest cell migration. Developmental

718

biology 344, 896-910.

719

Johnson, E., Seachrist, D. D., DeLeon-Rodriguez, C. M., Lozada, K. L., Miedler, J., Abdul-

720

Karim, F. W. and Keri, R. A. (2010). HER2/ErbB2-induced breast cancer cell migration and

721

invasion require p120 catenin activation of Rac1 and Cdc42. The Journal of biological chemistry

722

285, 29491-29501.

723

Koivusalo, M., Welch, C., Hayashi, H., Scott, C. C., Kim, M., Alexander, T., Touret, N.,

724

Hahn, K. M. and Grinstein, S. (2010). Amiloride inhibits macropinocytosis by lowering

725

submembranous pH and preventing Rac1 and Cdc42 signaling. The Journal of cell biology 188,

726

547-563.

727

Kowalczyk, A. P. and Nanes, B. A. (2012). Adherens junction turnover: regulating adhesion

728

through cadherin endocytosis, degradation, and recycling. Sub-cellular biochemistry 60, 197-

729

222.

730

Kulesa, P. M., Morrison, J. A. and Bailey, C. M. (2013). The neural crest and cancer: a

731

developmental spin on melanoma. Cells, tissues, organs 198, 12-21.

732

Le, T. L., Yap, A. S. and Stow, J. L. (1999). Recycling of E-cadherin: a potential mechanism

733

for regulating cadherin dynamics. The Journal of cell biology 146, 219-232.

734

Lee, R. T., Nagai, H., Nakaya, Y., Sheng, G., Trainor, P. A., Weston, J. A. and Thiery, J. P.

735

(2013). Cell delamination in the mesencephalic neural fold and its implication for the origin of

736

ectomesenchyme. Development 140, 4890-4902.

737

Levenberg, S., Yarden, A., Kam, Z. and Geiger, B. (1999). p27 is involved in N-cadherin-

738

mediated contact inhibition of cell growth and S-phase entry. Oncogene 18, 869-876.

739

Lim, J. and Thiery, J. P. (2012). Epithelial-mesenchymal transitions: insights from

740

development. Development 139, 3471-3486.

741

Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C. and Kirchhausen, T. (2006).

742

Dynasore, a cell-permeable inhibitor of dynamin. Developmental cell 10, 839-850.


743

McCusker, C. D. and Alfandari, D. (2009). Life after proteolysis: Exploring the signaling

744

capabilities of classical cadherin cleavage fragments. Communicative & integrative biology 2,

745

155-157.

746

Meng, W. and Takeichi, M. (2009). Adherens junction: molecular architecture and regulation.

747

Cold Spring Harbor perspectives in biology 1, a002899.

748

Mercer, J. and Helenius, A. (2008). Vaccinia virus uses macropinocytosis and apoptotic

749

mimicry to enter host cells. Science 320, 531-535.

750

Mercer, J. and Helenius, A. (2009). Virus entry by macropinocytosis. Nature cell biology 11,

751

510-520.

752

Miyashita, Y. and Ozawa, M. (2007a). A dileucine motif in its cytoplasmic domain directs

753

beta-catenin-uncoupled E-cadherin to the lysosome. Journal of cell science 120, 4395-4406.

754

Miyashita, Y. and Ozawa, M. (2007b). Increased internalization of p120-uncoupled E-cadherin

755

and a requirement for a dileucine motif in the cytoplasmic domain for endocytosis of the protein.

756

The Journal of biological chemistry 282, 11540-11548.

757

Nakagawa, S. and Takeichi, M. (1995). Neural crest cell-cell adhesion controlled by sequential

758

and subpopulation-specific expression of novel cadherins. Development 121, 1321-1332.

759

Nakagawa, S. and Takeichi, M. (1998). Neural crest emigration from the neural tube depends

760

on regulated cadherin expression. Development 125, 2963-2971.

761

Nanes, B. A., Chiasson-MacKenzie, C., Lowery, A. M., Ishiyama, N., Faundez, V., Ikura,

762

M., Vincent, P. A. and Kowalczyk, A. P. (2012). p120-catenin binding masks an endocytic

763

signal conserved in classical cadherins. The Journal of cell biology 199, 365-380.

764

Nieto, M. A. (2011). The ins and outs of the epithelial to mesenchymal transition in health and

765

disease. Annual review of cell and developmental biology 27, 347-376.

766

Orth, J. D., Krueger, E. W., Cao, H. and McNiven, M. A. (2002). The large GTPase dynamin

767

regulates actin comet formation and movement in living cells. Proceedings of the National

768

Academy of Sciences of the United States of America 99, 167-172.

769

Palacios, F., Schweitzer, J. K., Boshans, R. L. and D'Souza-Schorey, C. (2002). ARF6-GTP

770

recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions

771

disassembly. Nature cell biology 4, 929-936.


772

Palacios, F., Tushir, J. S., Fujita, Y. and D'Souza-Schorey, C. (2005). Lysosomal targeting of

773

E-cadherin: a unique mechanism for the down-regulation of cell-cell adhesion during epithelial

774

to mesenchymal transitions. Molecular and cellular biology 25, 389-402.

775

Paterson, A. D., Parton, R. G., Ferguson, C., Stow, J. L. and Yap, A. S. (2003).

776

Characterization of E-cadherin endocytosis in isolated MCF-7 and chinese hamster ovary cells:

777

the initial fate of unbound E-cadherin. The Journal of biological chemistry 278, 21050-21057.

778

Ridenour, D. A., McLennan, R., Teddy, J. M., Semerad, C. L., Haug, J. S. and Kulesa, P.

779

M. (2014). The neural crest cell cycle is related to phases of migration in the head. Development

780

141, 1095-1103.

781

Rogers, C. D., Saxena, A. and Bronner, M. E. (2013). Sip1 mediates an E-cadherin-to-N-

782

cadherin switch during cranial neural crest EMT. The Journal of cell biology 203, 835-847.

783

Schiffmacher, A. T., Padmanabhan, R., Jhingory, S. and Taneyhill, L. A. (2014). Cadherin-

784

6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural

785

crest. Molecular biology of the cell 25, 41-54.

786

Schnatwinkel, C., Christoforidis, S., Lindsay, M. R., Uttenweiler-Joseph, S., Wilm, M.,

787

Parton, R. G. and Zerial, M. (2004). The Rab5 effector Rabankyrin-5 regulates and coordinates

788

different endocytic mechanisms. PLoS biology 2, E261.

789

Sharma, M. and Henderson, B. R. (2007). IQ-domain GTPase-activating protein 1 regulates

790

beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and

791

adenomatous polyposis coli. The Journal of biological chemistry 282, 8545-8556.

792

Solis, G. P., Schrock, Y., Hulsbusch, N., Wiechers, M., Plattner, H. and Stuermer, C. A.

793

(2012). Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting

794

EGFR trafficking. Molecular biology of the cell 23, 1812-1825.

795

Spector, I., Shochet, N. R., Kashman, Y. and Groweiss, A. (1983). Latrunculins: novel marine

796

toxins that disrupt microfilament organization in cultured cells. Science 219, 493-495.

797

Swanson, J. A. (1989). Phorbol esters stimulate macropinocytosis and solute flow through

798

macrophages. Journal of cell science 94 ( Pt 1), 135-142.

799

Tai, C. Y., Mysore, S. P., Chiu, C. and Schuman, E. M. (2007). Activity-regulated N-cadherin

800

endocytosis. Neuron 54, 771-785.


801

Takai, Y., Ikeda, W., Ogita, H. and Rikitake, Y. (2008). The immunoglobulin-like cell

802

adhesion molecule nectin and its associated protein afadin. Annual review of cell and

803

developmental biology 24, 309-342.

804

Taneyhill, L. A. (2008). To adhere or not to adhere: the role of Cadherins in neural crest

805

development. Cell adhesion & migration 2, 223-230.

806

Taneyhill, L. A., Coles, E. G. and Bronner-Fraser, M. (2007). Snail2 directly represses

807

cadherin6B during epithelial-to-mesenchymal transitions of the neural crest. Development 134,

808

1481-1490.

809

Theveneau, E. and Mayor, R. (2012). Neural crest migration: interplay between

810

chemorepellents, chemoattractants, contact inhibition, epithelial-mesenchymal transition, and

811

collective cell migration. Wiley interdisciplinary reviews. Developmental biology 1, 435-445.

812

Thiery, J. P., Acloque, H., Huang, R. Y. and Nieto, M. A. (2009). Epithelial-mesenchymal

813

transitions in development and disease. Cell 139, 871-890.

814

Toyoshima, M., Tanaka, N., Aoki, J., Tanaka, Y., Murata, K., Kyuuma, M., Kobayashi, H.,

815

Ishii, N., Yaegashi, N. and Sugamura, K. (2007). Inhibition of tumor growth and metastasis by

816

depletion of vesicular sorting protein Hrs: its regulatory role on E-cadherin and beta-catenin.

817

Cancer research 67, 5162-5171.

818

Ulrich, F. and Heisenberg, C. P. (2009). Trafficking and cell migration. Traffic 10, 811-818.

819

Van Dyke, R. W. (2004). Heterotrimeric G protein subunits are located on rat liver endosomes.

820

BMC physiology 4, 1.

821

Wang, J. and Liu, X. J. (2003). A G protein-coupled receptor kinase induces Xenopus oocyte

822

maturation. The Journal of biological chemistry 278, 15809-15814.

823

Wu, C. Y., Jhingory, S. and Taneyhill, L. A. (2011). The tight junction scaffolding protein

824

cingulin regulates neural crest cell migration. Developmental dynamics : an official publication

825

of the American Association of Anatomists 240, 2309-2323.

826

Xiao, K., Allison, D. F., Buckley, K. M., Kottke, M. D., Vincent, P. A., Faundez, V. and

827

Kowalczyk, A. P. (2003a). Cellular levels of p120 catenin function as a set point for cadherin

828

expression levels in microvascular endothelial cells. The Journal of cell biology 163, 535-545.

829

Xiao, K., Allison, D. F., Kottke, M. D., Summers, S., Sorescu, G. P., Faundez, V. and

830

Kowalczyk, A. P. (2003b). Mechanisms of VE-cadherin processing and degradation in

831

microvascular endothelial cells. The Journal of biological chemistry 278, 19199-19208.

832

Xiao, K., Garner, J., Buckley, K. M., Vincent, P. A., Chiasson, C. M., Dejana, E., Faundez,

833

V. and Kowalczyk, A. P. (2005). p120-Catenin regulates clathrin-dependent endocytosis of VE-

834

cadherin. Molecular biology of the cell 16, 5141-5151.

835

Yanagisawa, M. and Anastasiadis, P. Z. (2006). p120 catenin is essential for mesenchymal

836

cadherin-mediated regulation of cell motility and invasiveness. The Journal of cell biology 174,

837

1087-1096.

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Accepted manuscript

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Figure Legends

843 844

Figure 1: Cad6B localizes to the cytoplasm. (A-A”) FlpIn-wtCad6B cells were fixed and

845

immunostained for Cad6B (green) and Na+K+ATPase (red). Panels represent the 3D composite

846

of several Z-stack images acquired with the confocal. Cad6B co-localizes with several

847

cytoplasmic puncta containing Na+K+ATPase (A”, arrowheads). (B-B”) Embryos possessing

848

neural crest cells initiating EMT were fixed and immunostained for Cad6B (green) and

849

Na+K+ATPase (red). Cad6B co-localizes with Na+K+ATPase-positive puncta (B”, arrowheads),

850

with membrane Cad6B denoted by arrows. (C-C’) Dorsal neural folds possessing premigratory

851

neural crest cells were explanted, allowed to undergo EMT, fixed, and immunostained for

852

Cad6B. (C’) is a higher magnification view of the boxed region in (C). Cad6B localizes to the

853

cytoplasm (C’, arrowheads). Scale bars: 10μm.

854 855

Figure 2: Cad6B puncta are endocytic rather than exocytic in nature. (A-A”) Cad6B

856

antibody recognizing the Cad6B extracellular domain (NT-6B) was introduced into the lumen of

857

embryos electroporated with membrane GFP, incubated to allow for EMT, fixed, and

858

immunostained for the NT-6B antibody (red) and GFP (green). (B-B”) High magnification

859

image of the boxed region in (A). Internalized NT-6B antibody-Cad6B complexes are seen in the

860

cytoplasm (B”, arrowheads) within the confines of GFP-expressing plasma membrane (B”,

861

arrow) in a delaminating neural crest cell. The NT-6B antibody was added to media of FlpIn-

862

wtCad6B cells (C-C”) and dorsal neural fold explants (D-D”), followed by incubation to allow

863

for EMT, a low pH buffer wash, fixation, and immunostaining for NT-6B (C, D) and HA (C’) or

864

CT-Ab (D’). Internalized NT-6B antibody-Cad6B complexes localize to the cytoplasm (C”, D”,

865

arrowheads), and Cad6B is still observed on the plasma membrane (C”, D”, arrows). All images

866

except (C-C’’’) represent the 3D composite of several Z-stack images acquired with the

867

confocal. Inset boxes in (C-C”, D-D”) show the original image, with the asterisks in (C) and (D)

868

indicating the location of the higher magnification field in the main panels. Scale bars: 10μm.


Accepted manuscript

869 870

Figure 3: Cad6B possesses putative endocytic motifs in its cytoplasmic domain. (A) A

871

portion of the alignment of the Cad6B juxtamembrane domain with several type II cadherins.

872

The dileucine and putative p120-catenin binding motif are highlighted (yellow). FlpIn cells

873

expressing wtCad6B (B-B”), LI645AA (C-C”), and EED666AAA (D-D”) were fixed and

874

immunostained for Cad6B (green) and p120-catenin (purple). Panels represent single confocal

875

plane images. Arrows point to membrane-bound Cad6B and p120-catenin, and carets indicate

876

Cad6B-positive, p120-catenin-negative cytoplasmic puncta. Inset boxes in (B-B”, C-C”, D-D”)

877

show the original image, with the asterisks in (B-D) indicating the location of the higher

878

magnification field in the main panels. Scale bars: 10μm.

879 880

Figure 4: Cad6B possess a functional endocytic motif. (A) Soluble and insoluble fractions

881

from FlpIn-wtCad6B, -LI645AA, and -EED666AAA cells were subjected to SDS-PAGE. (B)

882

Densitometric ratios of insoluble/soluble Cad6B from triplicate blots. (C) FlpIn cells expressing

883

Cad6B constructs were surface-biotinylated and incubated at 37˚C followed by biotin

884

immunoprecipitation with streptavidin-agarose and SDS-PAGE. (D) Quantification of

885

internalized biotinylated fraction (internalized biotinylated protein/total surface biotinylated

886

protein). (E) FlpIn cells expressing Cad6B constructs were treated with cycloheximide (CHX),

887

lysed, and subjected to SDS-PAGE. Irrelevant lanes between time 0 and the other two time

888

points were cropped out of the LI645AA immunoblot. (F) Quantification of the densitometric

889

ratios (Cad6B/actin) from (E). Error bars represent ± S.E.M, and an asterisk denotes a

890

statistically significant difference.

891


892

Figure 5: Cad6B does not co-localize with caveolin-1 but partially co-localizes with clathrin

893

in vitro. (A-A”) FlpInwtCad6B cells were fixed and immunostained for Cad6B (green) and

894

caveolin-1 (red). Caret shows absence Cad6B and caveolin-1 co-localization. FlpIn cells

895

expressing wtCad6B (B-B”), LI645AA (C-C”), and EED666AAA (D-D”) were fixed, and

896

immunostaining was performed for Cad6B (green) and clathrin (red). Panels represent single

897

confocal plane images. Arrowheads in (B”, C”, D”) show Cad6B and clathrin co-localization,

898

and carets point to Cad6B puncta not co-localizing with clathrin. Inset boxes show the original

899

image, with the asterisks in (A-D) indicating the location of the higher magnification field in the

900

main panels. Scale bars: 10μm.

901 902

Figure 6: Cad6B partially co-localizes with clathrin in vivo. (A-A”) Embryos in which neural

903

crest cells are actively undergoing EMT were fixed and immunostained for Cad6B (green) and

904

clathrin (red). (B-B”) is a higher magnification view of the boxed region in (A). Panels represent

905

the 3D composite of several Z-stack images acquired with the confocal. Arrowheads in (B”)

906

indicate Cad6B and clathrin co-localization, and carets show Cad6B-positive, clathrin-negative

907

puncta. Scale bars: 10μm.

908 909

Figure 7: Cad6B partially co-localizes with p120-catenin in cytosolic puncta in vivo. (A-A”)

910

Embryos in which neural crest cells are actively undergoing EMT were fixed and immunostained

911

for Cad6B (green) and p120-catenin (red). (B-B”) is a higher magnification view of the boxed

912

region in (A). Panels represent the 3D composite of several Z-stack images acquired with the

913

confocal. Arrowheads in (B”) point to co-localized Cad6B and p120-catenin, and carets show

914

Cad6B-positive, p120-catenin-negative puncta. Scale bars: 10μm.

915 916

Figure 8: Cad6B undergoes dynamin-dependent macropinocytosis. Vehicle (DMSO, A-A”),

917

Dynasore (B-B”), Latrunculin A (C-C”), or EIPA (D-D”) was added to the explant media.

918

Explants were incubated to allow for EMT, fixed, and immunostained for Cad6B (green) and

919

p120-catenin (red: A’, A”, B’, B”, D’, D”) or stained with phalloidin (red: C’, C”). Arrows and

920

arrowhead in (A”) represent Cad6B and p120-catenin co-staining at the membrane and

921

cytoplasm, respectively. Arrowheads in (B”, D”) show large Cad6B and p120-catenin double-

922

positive cytoplasmic puncta. Arrows in (C”) indicate membrane Cad6B. Carets in (A”, B”, D”)

923

point to Cad6B-positive, p120-catenin-negative puncta. Panels represent the 3D composite of

924

several Z-stack images acquired with the confocal. Inset boxes show the original image, with the

925

asterisks in (A-D) indicating the location of the higher magnification field in the main panels.

926

Scale bars: 10μm.

927 928


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929


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Accepted manuscript

FoxD3 regulates cranial neural crest EMT via downregulation of tetraspanin18 independent of its functions during neural crest formation.

Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT.

Cell death in cranial neural crest development.

The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis.

Cell cycle changes during neural crest cell differentiation in vitro.

Expression and function of transcription factor cMyb during cranial neural crest development.

Neural crest and placode interaction during the development of the cranial sensory system.

SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination.

Neural crest cell signaling pathways critical to cranial bone development and pathology.

Chemotaxis during neural crest migration.

Cadherin-6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural crest.

Dataset of TWIST1-regulated genes in the cranial mesoderm and a transcriptome comparison of cranial mesoderm and cranial neural crest.

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish.

Migration of cranial neural crest cells in a cell-free hyaluronate-rich matrix.

Expression of cell adhesion molecules during initiation and cessation of neural crest cell migration.

Rabconnectin-3a regulates vesicle endocytosis and canonical Wnt signaling in zebrafish neural crest migration.

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo.

Modelling collective cell migration of neural crest.

Glial cell lineages in the neural crest.

A catecholaminergic sensory neuron phenotype in cranial derivatives of the neural crest: regulation by cell aggregation and nerve growth factor.

Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos.

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

Cranial neural crest deletion of VEGFa causes cleft palate with aberrant vascular and bone development.

Comparison of the Odontogenic Differentiation Potential of Dental Follicle, Dental Papilla, and Cranial Neural Crest Cells.