Lgr4 and Lgr5 drive the formation of long actin-rich ... · Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions Joshua C. Snyder1, Lauren K. Rochelle1,Se´bastien

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RESEARCH ARTICLE

Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-likemembrane protrusions

Joshua C. Snyder1, Lauren K. Rochelle1, Sebastien Marion1, H. Kim Lyerly2, Larry S. Barak1 andMarc G. Caron1,*

ABSTRACT

Embryonic development and adult tissue homeostasis require

precise information exchange between cells and their

microenvironment to coordinate cell behavior. A specialized class

of ultra-long actin-rich filopodia, termed cytonemes, provides one

mechanism for this spatiotemporal regulation of extracellular cues.

We provide here a mechanism whereby the stem-cell marker Lgr5,

and its family member Lgr4, promote the formation of cytonemes.

Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 mm, are

generated through stabilization of nascent filopodia from an

underlying lamellipodial-like network and functionally provide a

pipeline for the transit of signaling effectors. As proof-of-principle,

we demonstrate that Lgr5-induced cytonemes act as conduits for

cell signaling by demonstrating that the actin motor and filopodial

cargo carrier protein myosin X (Myo10) and the G-protein-coupled

receptor (GPCR) signaling effector b-arrestin-2 (Arrb2) transit into

cytonemes. This work delineates a biological function for Lgr4 and

Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5

function and cytonemes in mammalian stem cell and cancer stem

cell behavior.

KEY WORDS: Lgr5, GPCR, Cytoneme, Stem cell

INTRODUCTIONThe G-protein-coupled receptor (GPCR) Lgr5 genetically

identifies a population of intestinal crypt stem cells (Barker

et al., 2007). Since this observation, the ability to distinguish stem

cells in other tissues on the basis of Lgr5 expression has proved to

be a successful strategy in the stomach (Barker et al., 2010), skin

(Jaks et al., 2008), liver (Huch et al., 2013b), pancreas (Huch

et al., 2013a), kidney (Barker et al., 2012), mammary gland (de

Visser et al., 2012; Plaks et al., 2013) and developing

hematopoietic system (Liu et al., 2014). Lgr5 is the founding

member of a subclass of the leucine-rich G-protein-coupled

receptors (Lgrs) that include Lgr4 and Lgr6 (Hsu et al., 2000; Hsu

et al., 1998). In addition to Lgr5, Lgr4 and Lgr6 are also used as

markers of the stem and progenitor cell lineages within tissues.

Lgr4 expression identifies an expanded pool of progenitor cells in

the intestinal crypt, which also includes those cells expressing

Lgr5 (de Lau et al., 2011). Lgr6 expression identifies a more

primordial stem cell with restricted cellular expression compared

with Lgr5-expressing hair follicle stem cells (Snippert et al.,

2010). The clinical relevance of these discoveries has been

underscored by the fact that Lgr5-expressing cells possess greater

tumorigenic potential than their differentiated progeny (Barker

et al., 2010; Barker et al., 2009), and the demonstration that Lgr5-

expressing cells in intestinal adenomas are cancer stem cells

(Schepers et al., 2012).

The complement of membrane receptors on stem cells

might confer them with intrinsic regulatory capacity by tightly

controlling their response to extracellular cues. Lgr4–6 signal by

a non-canonical G-protein-independent mechanism by binding

to R-spondins (Carmon et al., 2012; de Lau et al., 2011) or

Norrin (Deng et al., 2013) and potentiating Wnt–bcatenin signal

transduction. However, it is likely that other signaling pathways

and biological processes are engaged by Lgr4–6. In fact, GPCR

signaling is multifaceted in nature and is tightly regulated by

endocytosis of activated receptors (Rajagopal et al., 2010). We

have reported on the cellular trafficking properties of Lgr5 using

EGFP-tagged forms of the receptor and found that the C-terminal

tail regulates its unique constitutive internalization and retrograde

trafficking to the trans-Golgi network in the apparent absence of

ligand (Snyder et al., 2013b). Therefore, we hypothesized that

inhibiting this constitutive internalization would provide new

insight into the cellular function of Lgr5. Surprisingly, we

discovered that the plasma membrane expression of Lgr5 and

Lgr4 provide a mechanism for driving the formation of ultra-long

actin-rich membrane protrusions that reach lengths approaching

80 mm. These filopodia are phenotypically and molecularly

similar to a specialized class of filopodia previously described

by Kornberg in the Drosophila wing imaginal disc as ‘cell

threads’ or cytonemes (Ramırez-Weber and Kornberg, 1999).

Filopodia are typically of modest length (often ,10 mm)

owing to the biophysical forces required to deform the plasma

membrane (Mogilner and Rubinstein, 2005). By contrast, ultra-

long actin-rich filopodia were first observed in sea urchin

embryogenesis by Wolpert in 1961 (Gustafson, 1963) and

further investigated by McClay in 1995 (Miller et al., 1995).

Since their initial description, these structures have been named

cytonemes to distinguish them from shorter filopodia. Cytonemes

have many distinctive characteristics including their fragility,

width and length; all features shared by Lgr4 and Lgr5-

induced protrusions. Cytonemes can compartmentalize signaling

pathways (Roy et al., 2011) and direct the transfer of morphogens

between cells (Roy et al., 2014). In Drosophila, cytonemes

orient towards a morphogen-producing cell or emanate from a

morphogen-producing cell towards a target cell. Recent studies

in Drosophila indicate that cytonemes provide a platform for

transmitting morphogens in the stem cell niche of the Drosophila

germarium (Rojas-Rıos et al., 2012). Therefore, cytonemes can

1Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,USA. 2Department of Surgery, Duke University Medical Center, Durham, NC27710, USA.

*Author for correspondence ([email protected])

Received 18 November 2014; Accepted 27 January 2015

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1230–1240 doi:10.1242/jcs.166322

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be employed to exquisitely regulate the scope and precision ofsignaling during tissue development and maintenance.

Despite these elegant studies, only a few reports haveinvestigated similar structures in mammalian cells and, untilrecently, even effectors responsible for their formation inDrosophila were missing entirely (Roy et al., 2014). Therefore,

questions regarding the mechanisms that drive cytonemeformation and their utility in signaling remain largelyunexplored in mammalian systems (Affolter and Basler, 2011;

Kornberg and Roy, 2014). Our study answers these questions. Wedemonstrate that Lgr5 and Lgr4 provide a receptor-basedmechanism for triggering the formation of cytonemes and

further illustrate that these cytonemes can be scaffolds forsignaling effectors in a mammalian cell system. These findingssuggest that stem cells might possess the hardware for regulating

signaling at a distance.

RESULTSLgr5 and Lgr4 expression in mammalian cells induces therobust formation of membrane protrusionsNormally, Lgr5 is constitutively internalized, resulting inintracellular localization of EGFP-tagged receptor (Fig. 1A–C)(Snyder et al., 2013b). However, when the internalization of Lgr5

was blocked either by overexpression of dominant-negativedynamin-1 (K44A), by C-terminal tail truncation at position834 (834del) or by exchanging the C-terminal tail for that of

the human vasopressin V2 receptor (V2R; Lgr5–V2Rtail)(Fig. 1A,B,D–F), we discovered the robust formation ofmembrane protrusions at the interface between the cell surface

and the substrate by confocal microscopy (Fig. 1H–J). Unlikewild-type Lgr5, wild-type Lgr4 was expressed more robustly atthe plasma membrane and cells transfected with wild-type Lgr4

displayed extensive protrusions (Fig. 1A,B,G,K). Lgr4 and Lgr5

Fig. 1. Cell-surface expression of Lgr5 or Lgr4 induces the robust formation of membrane protrusions. (A) An on-cell enzyme-linked immunosorbent assay(ELISA) was performed to measure the membrane (live cell stained) and total (fixed, permeabilized and stained) expression of each receptor in HEK cells. Theimages shown are representative of more than three experiments. (B) Relative membrane expression was determined by calculating the ratio of membrane tototal expression values for each receptor and then normalizing to Lgr5FL across multiple experiments. The data show the mean6s.e.m.; *P,0.05 (one-way ANOVAwith Bonferroni post-hoc testing). Confocal optical sections of live cells transiently expressing (C) Lgr5FL, (D) Lgr5FL + dynamin-1 K44A (K44A), (E) 834del,(F) Lgr5–V2Rtail (Lgr5/V2Rtail) or (G) Lgr4 to determine their expression at the plasma membrane. HEK cells were transiently transfected with EGFP-tagged(H) Lgr5FL+K44A, (I) 834del, (J) Lgr5–V2Rtail or (K) Lgr4, imaged live on a confocal microscope and rendered in three dimensions. Scale bars: 10 mm.

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membrane protrusions are very fragile, and can be severed byover-fixation or even by mild physical perturbations. These

structures were often found to be directed towards the basolateralsurface and branched upon their contact with the substratum(supplementary material Movie 1). These data demonstrate thatmembrane protrusions are coincident with the membrane

expression for each receptor.We next tested the hypothesis that this observation is a

hallmark of Lgr signaling and not a broader and previously

undocumented characteristic of GPCR expression. We transientlytransfected cells with 11 EGFP or EYFP-tagged GPCRs, inaddition to variants of Lgr5 or Lgr4, and imaged for the presence

of membrane protrusions. Cells were blindly scored and thencategorized as either an Lgr family member or not based upontheir ability to form membrane protrusions. We successfully

determined that, as opposed to all other GPCRs tested, allmembrane-localized forms of Lgr5 or Lgr4 were competent toform these long thin actin filaments (Fig. 2). Relative to Lgr5,receptors like the b2-AR (ADRB2) do not possess considerable

constitutive internalization properties, illustrating that simplypreventing GPCR internalization does not cause the formationof membrane protrusions (Snyder et al., 2013b). However,

to completely discount the off-target effects of blockingendocytosis, the b2-AR was co-expressed with dynamin K44A.Importantly, no morphological changes were found (Fig. 2P).

These data demonstrate that the dramatic induction of membraneprotrusions are a specific consequence of overexpressing Lgr4 orLgr5 at the plasma membrane.

Morphological and biochemical characterization of Lgr4- andLgr5-induced membrane protrusionsThe EGFP tag on the receptor enabled a remarkable visualizationof the membrane protrusions. These structures go unnoticed if thereceptors are not tagged or can be broken if non-optimal fixationand antibody staining is performed instead of live imaging for

EGFP. The membrane protrusions formed gave the appearance offilopodia-like structures that normally comprised polymerized F-actin. Therefore, we tested whether Lgr-induced membrane

protrusions are F-actin positive and whether these F-actinstructures can be induced by a control GPCR. We performed F-actin staining for each of the Lgr5 or Lgr4-EGFP constructs and

compared this to that of non-transfected HEK cells and thoseexpressing human b2-AR. As would be expected from Fig. 2,expression of b2-AR does not induce significant F-actin

membrane protrusions (Fig. 3A) nor are these protrusionsinduced in non-transfected HEK cells (Fig. 3B, asterisk),according to previously published observations (Lin et al.,2007). In previous experiments that utilized GFP to visualize

membrane protrusions, we did not notice any apparent membraneprotrusions for wild-type full-length Lgr5 (Lgr5FL). However,owing to its labile cell surface expression, GFP expression at the

membrane is below the limits of detection. Therefore, wehypothesized that Lgr5FL could induce similar membraneprotrusions if instead they were visualized through F-actin

staining. As expected, expression of Lgr5FL also inducedmembrane protrusions, thereby verifying that this is a bona fideproperty of this receptor when expressed in its wild-type form

Fig. 2. Selective generation of membraneprotrusions through expression of Lgr4 orLgr5 and not other GPCRs. (A–P) Theindicated EGFP- or EYFP-tagged GPCRs weretransiently transfected into cells that were thenconfocal imaged and scored blindly. Theimages were scored for the presence orabsence of membrane protrusions. Only Lgr5-or Lgr4-transfected cells were scored as havingformed extensive protrusions. The presentedimages are three-dimensionally rendered from aseries of optical slices to facilitate coincidentviewing of both the apical and basolateralplasma membrane. (A) AVRP2, vasopressin-2receptor; (B) SMO, smoothened; (C) GHSR,growth hormone secretagogue receptor;(D) ADRB2, b2-adrenergic receptor;(E) ADRA1A-opto, a1-adrenergic receptor–optogenetic chimera (Airan et al., 2009);(F) CNR1, cannabinoid receptor 1; (G) AGTR1,angiotensin II receptor type 1; (H) AGTR2,angiotensin II receptor type 2; (I) NTSR1,neurotensin receptor 1; (J) D2R, dopaminereceptor D2; (K) Lgr5FL, (L) Lgr4, (M)Lgr5FL+K44A, (N) Lgr5834del, (O) Lgr5–V2Rtail(Lgr5/V2Rtail), (P) ADRB2:b2-adrenergicreceptor+K44A. The latter construct wastransfected into cells by using Genecellin. Scalebars: 10 mm.

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(Fig. 3B). We also conducted F-actin staining for Lgr5FL that wasstabilized on the surface through K44A overexpression or byalterations to its C-terminus. We confirmed that expression of

each of these isoforms resulted in F-actin-positive membraneprotrusions (Fig. 3C–E). Likewise, Lgr4-induced protrusions alsoshared this property (Fig. 3F). To test the ability to form actin-

rich membrane protrusions in an unbiased manner and furtherdescribe this unique property of Lgr proteins, we performed ablinded and quantitative analysis of their number (Fig. 4A) and

length (Fig. 4B). These data demonstrated that non-transfectedcells and b2-AR-transfected ones have similarly few and shortactin-rich membrane protrusions. This is in contrast to cellstransfected with Lgr5FL, its membrane stabilized forms and Lgr4,

which all drive an increase in the number and length of membraneprotrusions. Further analysis also demonstrated the width of these

structures to be ,0.53 mm (60.019, 6s.e.m.) – consistent with afilopodium-like structure (Fig. 4C,D) (Sheetz et al., 1992).

To further characterize Lgr-induced membrane protrusions, weprobed for their ability to incorporate other stereotypical filopodia

components. Indeed, both 834del and Lgr5–V2Rtail receptor-induced membrane protrusions can incorporate actin effectors,including the anti-capping protein VASP and the actin-bundling

protein fascin (Fig. 5A,B) (Svitkina et al., 2003). In contrast tofascin, which is present homogeneously throughout theprotrusion, VASP can be found concentrated at the tip, as

published previously (Mejillano et al., 2004). These resultsdemonstrate that Lgr5-induced membrane protrusions sharesimilar molecular features to filopodia yet remain unique,

owing to their great lengths.

Formation of Lgr5-induced membrane protrusions and theircharacterization as cytonemesSimilar to filopodia, Lgr5-induced protrusions are sensitive tocytochalasin B (Burnette et al., 2007). Overnight treatment with10 mM cytochalasin B results in total ablation of Lgr5-induced

protrusions (Fig. 5C, inset). The majority of Lgr5-inducedprotrusions are rapidly remodeled, consolidated and ablated in,5 hours of cytochalasin B treatment (Fig. 5C; supplementary

material Movie 2). Finally, upon cytochalasin B washout, cellsrapidly restore their membrane protrusions within 1 hour by amechanism that resembles filopodium formation from an

underlying Arp2/3 lamellipodial-like interfilopodial veil asdescribed previously (Fig. 5D; supplementary material Movie 3;Korobova and Svitkina, 2008). Next, the formation of Lgr5-induced membrane protrusions in untreated cells was studied in a

more focused timecourse. Similar to results observed followingcytochalasin washout, the formation of Lgr5-positive membraneprotrusions in untreated cells also appeared to occur through

a rapid remodeling of a pre-existing lamellipodial-likeinterfilopodial veil (Fig. 6A; supplementary material Movie 4).De novo branching of Lgr5-induced membrane protrusions was

never observed. Rather, fully branched Lgr5-positive membraneprotrusions emerged from the underlying lamellipodial-likenetwork. Fully mature Lgr5-induced cytonemes appeared to berelatively static structures (Fig. 6A, asterisk), whereas nascent

protrusions were observed to dynamically elongate and retract,closely resembling the dynamic nature of filopodia (Svitkinaet al., 2003). In some instances, this elongation was followed

by retraction of the lamellipodial-like interfilopodial veiland stabilization of an Lgr5-induced filopodium (Fig. 6A,arrowhead). In other cases, the elongation was immediately

followed by retraction and absorption by the plasma membrane(Fig. 6A, arrow). These data indicate that simultaneouselongation of filopodia and dissolution of the lamellipodial-like

interfilopodial veil enables the formation of highly elongatedand branched Lgr5-induced membrane protrusions with greatrapidity.

Previous literature has demonstrated that ultra-long and actin-

rich membrane protrusions are a specialized class of actin-richfilopodia termed cytonemes. Cytonemes are biochemically andmorphologically similar to Lgr4- and Lgr5-induced membrane

protrusions and are also sensitive to cytochalasin treatment(Hsiung et al., 2005; Ramırez-Weber and Kornberg, 1999). Thelive imaging results suggest that that Lgr4- and Lgr5-induced

membrane protrusions most closely resemble cytonemes.

Fig. 3. Lgr4- and Lgr5-induced membrane protrusions are rich inpolymerized actin. Actin staining (red, left column) in HEK cells transientlyexpressing the indicated GFP-tagged constructs (green, middle column) wasperformed for (A) b2-AR, (B) Lgr5FL (the asterisk denotes a non-transfectedcell), (C) Lgr5FL+K44A, (D) Lgr5–V2Rtail (Lgr5/V2Rtail), (E) 834del and(F) Lgr4. Three-dimensional reconstructed images were created from Z-stack scans for each cell. Multiple cells across multiple experiments wereimaged. Imaging was performed blinded for subsequent utilization inmorphometric analysis of protrusion number and length. Scale bar: 10 mm.

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However, markers that distinguish cytonemes from other longactin-rich protrusions, such as retraction fibers, do not exist

and, as such, alternative classifications cannot be completelydiscounted.

Lgr5-induced cytonemes as cell signaling platformsBecause cytonemes are well-known cell signaling centers, wetested whether Lgr5-induced cytonemes could serve as a supportplatform for cell signal transduction. Their great lengths suggested

that signaling effectors might require a transport mechanism toreach the extreme distal tips of each membrane protrusion. We thustested the candidate carrier molecule myosin X (MyoX, also

known as Myo10). Myosin X is an actin motor and unconventionalmyosin, previously shown to travel along actin-rich filopodia anddeliver cargo to filopodia tips (Berg and Cheney, 2002; Zhang

et al., 2004). Indeed, MyoX can travel along Lgr5-inducedcytonemes (Fig. 6B; supplementary material Movie 5). We nextsought to establish conditions to test whether Lgr-induced

cytonemes could scaffold cell signaling molecules. As a modelcell signaling system we focused on GPCR signaling, specifically,the ability of b2-AR to recruit the adaptor protein b-arrestin-2(barr2, also known as Arrb2) upon receiving a ligand-mediated

stimulus. Translocation of barr2 is a hallmark of prototypicalGPCR signaling and receptor desensitization, and the protein actsas a scaffold for an array of downstream signaling cascades (Barak

et al., 1997; Rajagopal et al., 2010). First, we tested whether b2-ARcould access cytonemes. We found that, despite the inability of b2-

AR to form cytonemes, it localized to them when co-expressedwith Lgr5 (Fig. 7A). Next, we tested whether barr2 could travelalong Lgr-induced cytonemes. Surprisingly, barr2–EGFP was not

detected in the Lgr5-induced cytonemes (Fig. 7B). However,following agonist-mediated stimulation of co-expressed b2-AR,we found rapid recruitment of barr2 throughout the Lgr-inducedcytoneme, in addition to its typical plasma membrane distribution

described previously (Fig. 7C,D) (Barak et al., 1997). The rapidentry of barr2 into the Lgr-induced cytoneme suggested theexistence of a gating system for enabling barr2 translocation into

Lgr-induced protrusions. Indeed, we were able to demonstrate aninteraction between MyoX and barr2 using in vitro GST pulldowns(Fig. 8A,B). This interaction occurred in vitro both before and after

the stimulus, as found through co-immunoprecipitation of MyoXwith barr2 (Fig. 8C). These data demonstrate that Lgr-inducedcytonemes harbor the components necessary to serve as a platform

for delivery of signaling effectors, and they provide one plausiblemechanism whereby signaling effectors might transit intocytonemes.

DISCUSSIONLgr4 and Lgr5 are robust markers of the stem cell hierarchy buttheir function in cells has remained mysterious. We provide cell

Fig. 4. Morphometric analysis of Lgr4- and Lgr5-induced cytonemes. The number and length of F-actin-positive membrane protrusions in HEK cells thatwere non-transfected (NT) or transiently expressing b2-AR, Lgr5FL, Lgr5FL + K44A, Lgr5–V2Rtail (Lgr5/V2Rtail), 834del and Lgr4 were counted and arerepresented in (A) as the total number of protrusions per 10 mm. Data show the mean6s.e.m.; *P,0.05 (one-way ANOVA with Bonferroni post-hoc testing). Thedata are presented graphically in B to comparatively show the length and number of membrane protrusions as a function of the transiently expressed receptor.(C) HEK cells were transiently transfected with 834del–GFP and imaged live at 1006. A 12-mm line was drawn perpendicular to several protrusions andfluorescence intensity is plotted in D. (D) Pixel intensity as a function of line distance (from left to right in C). These data were imported into GraphPad Prism andfitted with a Gaussian curve, and the width of the cytoneme was calculated by determining the width of the Gaussian curve. A total of 17 membrane protrusionsfrom two different cells were used to calculate the mean width of Lgr5-induced membrane protrusions [0.53 mm; 60.019 (s.e.m.)].

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biological evidence for their roles as robust drivers of actin-richmembrane protrusions termed cytonemes. Cytoneme number andlength are dependent upon the expression of Lgr4 and Lgr5 at the

cell surface. Lgr4- and Lgr5-induced cytonemes are actin-dependent and contain typical actin-modifier proteins such asVASP and fascin. Their formation occurs through concerted

stabilization of nascent filopodia and retraction of the cellmembrane. Lgr5-induced cytonemes play a role in cell signalingby providing a conduit for the unconventional myosin motor

protein MyoX and its delivery of activated signaling effectors tothe tip of an activated cytoneme.

In 1924, Spemann and Mangold first alluded to the presence of

what are now referred to as morphogen gradients (De Robertis,2006). In the years since, this paradigm has provided a foundationfor modern day developmental and stem cell biology by aiding inthe understanding of the prototypical morphogens Wnt, Shh and

BMP, and their roles in cell fate. However, the mechanismwhereby gradients are established for these morphogens remainselusive. Many morphogens, Wnt and Shh included, undergo

significant post-translational lipid and cholesterol modifications,deeming these secreted proteins highly insoluble and membraneassociated. Many postulates have been formed in order to

explain how gradients could be formed with insoluble proteins,including delivery by ultra-long filopodia termed cytonemes, asdemonstrated in Drosophila (Port and Basler, 2010). Therefore,in a similar manner, Lgr4- or Lgr5-driven cytonemes could

provide a mechanism for the delivery of morphogens to amammalian stem cell niche.

Recent evidence suggests that intestinal Wnt is not provided by

the epithelium but most likely by cells from the underlyingmesenchyme (Kabiri et al., 2014). Therefore, epithelium-to-mesenchyme projections could provide an avenue for the transit

of insoluble Wnts through a dense layer of extracellular matrix andbasement membrane. Drosophila Lgr2 is the ortholog to Lgr4–6and, in contrast to Lgr4 and Lgr5, has a well-defined G-protein-

dependent signaling pathway. Lgr2 binds to its cognateheterodimeric hormone ligand Bursicon (Burs or Pburs) andstrongly couples to G proteins to stimulate cAMP production. In a

recent study, long-distance transmission of Bursicon to Lgr2 hasbeen postulated to indirectly regulate the intestinal stem cell niche(Scopelliti et al., 2014). These data indirectly suggest that Lgrproteins in general might actually utilize cytonemes to regulate the

reception of their cognate ligands. Our study provides evidence forLgr4- and Lgr5-driven cytoneme formation and provides onemechanism whereby directional and compartmentalized signaling

in mammalian stem cells could be regulated.There are two tantalizing reports that might begin to confirm

our in vitro findings in mouse model systems. First, Lgr5-positive

cells, when observed at the ultrastructural level, possess ‘‘slenderextensions of apical cytoplasm’’ in vivo (Barker et al., 2007).Second, mice deficient for Lgr4 have an eyelid closure defect thatis coincident with reduced filopodia number and loss of growth

Fig. 5. Biochemical and functional characterization of Lgr5-induced cytonemes. (A) VASP–GFP (green) or (B) fascin–GFP (green) were cotransfected intocells with 834del- (upper panel) or Lgr5–V2Rtail (Lgr5/V2Rtail, lower panel) HA-tagged receptors (antibody stained in red), and the cells were confocal imaged.The data are presented as single-channel images and merged images. VASP is present throughout the entire cytoneme but can concentrate at the tip (arrows).(C) Overnight treatment with 10 mM cytochalasin B ablates 834del-induced cytonemes (inset). 834del-expressing cells were treated with 10 mM cytochalasin Band imaged every 30 minutes for 5 hours (see supplementary material Movie 2 for full timecourse). The asterisk denotes a cytoneme that remains aftertreatment; the dotted region demonstrates consolidation and ablation of a cluster of cytonemes; the arrowhead depicts rapid loss of shorter cytonemes.(D) 834del-expressing cells were treated with 10 mM cytochalasin B overnight, washed five times and then imaged every 30 minutes for 2.5 hours (seesupplementary material Movie 3 for full timecourse). The arrow denotes rapid appearance of cytonemes from an existing lamellipodial-like framework. Scale bar:10 mm.

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factor signaling in the eyelid (Jin et al., 2008). Additionally,conditional deletion of Lgr4 and Lgr5 in adult intestinal stemcells results in marked loss of intestinal crypts. In adults, deletion

of Lgr4 or Lgr5 alone has only modest effects, suggesting theexistence of a compensatory mechanism provided by eachreceptor (de Lau et al., 2011) that also exists during

development (Kinzel et al., 2014). Taken together, these datapoint to an important regulatory role for Lgr4 and Lgr5 in vivo

that might be the result of cytoneme-based cell signaling.

Lgr4–6 are evolutionarily conserved along with thyroidstimulating hormone receptor (TSHR), luteinizing hormonereceptor (LHR) and follicle-stimulating hormone receptor(FSHR), and are likewise marked by seven-transmembrane

domains and a uniquely large N-terminal ectodomain composedof leucine-rich repeats. In contrast to TSHR, LHR and FSHR, thesignaling cascade that Lgr4–6 modulates is only recently being

elucidated and, surprisingly, does not yet include ligands that arecapable of coupling Lgr4–6 to G protein or b-arrestins (Carmonet al., 2011; de Lau et al., 1998; Deng et al., 2013). While G-

protein-coupling or b-arrestin translocation to Lgr4–6 has notbeen demonstrated, circumstantial evidence is mounting tosuggest that these signaling effectors can be engaged and that

the ligands and effectors capable of eliciting this behavior remainunknown. Lgr5 possesses b-arrestin recruitment motifs that arefully active under the appropriate conditions (Snyder et al.,2013a). Recent evidence demonstrates that the cerebellar Purkinje

cells of hypomorphic Lgr4 mice have reduced levels of phospho-CREB, which can be restored through treatment with forskolin(Guan et al., 2014). This is further evidence that these receptors

might couple to G proteins. These data together illustrate that thisfamily of receptors remains highly enigmatic, but point to thepossible role for other ligands that might signal through Lgr4 and

Lgr5 and, ultimately, regulate cytoneme formation. Interestingly,recent work has shown that the R-spondin–Lgr4 signaling axismight also converge on actin (Carmon et al., 2014) and suggests

that Lgr4 and Lgr5 are uniquely primed for regulating thecytoskeleton. From our experience with GPCRs and the datashown in Fig. 2, this behavior does appear to be highly specific

for Lgr4 and Lgr5. However, in a recent report, expression andactivation of the GPCR A3-adenosine receptor (A3AR) was alsofound to initiate the formation of cytonemes in human neutrophilsfor use in scavenging bacteria (Corriden et al., 2013). In contrast

to Lgr4- and Lgr5-induced cytonemes, A3AR-induced cytonemesappear to be distinct based upon the modest number formed percell and their inability to branch. At this time, further studies

will be necessary to distinguish the similarities and differencesbetween these structures. We are unable to determine whethercytonemes can be formed by Lgr6 as we are unable to express this

receptor at the plasma membrane. Lgr4–6 are at the crossroads ofstem cell biology and cancer. Therefore, elucidating the cellularand biological function of this receptor family is essential to

unlock their potential as new drug targets.Invadopodia are a class of filopodia that are similar to

cytonemes. These structures are found in invasive cancer cellsand are thought to promote cellular invasion through digestion of

extracellular matrix and underlying mesenchyme (Chen, 1989;Linder, 2007). Among the many similarities to Lgr5 cytonemes,invadopodia are also characterized by expression of VASP, fascin

Fig. 6. Formation of Lgr5-induced cytonemes and transit of motor proteins. (A) Confocal images (1006) of HEK cells transiently transfected with 834del–GFP. The cells were imaged for 30.9 minutes. Nascent protrusions rapidly elongate and retract by 10.5 minutes (arrow) or stabilize and form cytonemes uponretraction of the cell membrane (arrowhead). The asterisk shows a static Lgr5-induced cytoneme. See supplementary material Movie 4. (B) Strawberry-tagged834del (red) and GFP-tagged myosin X (green) were imaged for 30.9 minutes. Myosin X travels from the distal tip of the membrane protrusion and israpidly transported in a retrograde fashion to the cell body by 10.5 minutes (arrowhead) or travels from the membrane protrusion anterograde to the distal tip by6.3 min, undergoes retrograde transport to the cell body by 16.7 min, and finally anterograde transport from the cell body towards the distal tip at 23.0 min(asterisk). See supplementary material Movie 5. Scale bars: 10 mm.

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and MyoX (Schoumacher et al., 2010). Interestingly, MyoX isable to induce filopodia and delivers integrins to the cell surface

to increase cell adhesion (Berg and Cheney, 2002; Zhang et al.,2004). Two recent reports suggest that MyoX is an importantmarker of cancer invadopodia and cancer cell invasiveness.

MyoX-dependent transport of integrins to the filopodium tip isessential for tumor cell invasion (Arjonen et al., 2014) andmetastasis (Cao et al., 2014). These reports also demonstrate thatMyoX expression is an important marker of aggressive and highly

malignant breast cancers. We already know that Lgr5 cellsrepresent the cell of origin in gastrointestinal cancers (Barkeret al., 2009; Schepers et al., 2012). Therefore, our data suggests a

potentially insidious role for cytonemes in carcinogenesis,whereby they are hijacked and exploited for cancer cellsurvival, invasion and metastasis. Our study provides the basis

for further characterizing Lgr4 and Lgr5 in vivo. The associationof Lgr5 and Lgr4 with stem cells, their remarkable ability toinduce cytonemes in vitro, and the proposed role for cytonemes inmorphogen-mediated stem cell signaling, provide a previously

unrealized opportunity for future studies seeking to define rolesfor these receptors in normal tissue homeostasis and cancer.

MATERIALS AND METHODSPlasmids, cell lines and transfectionClones encoding GPCRs were either previously described [Lgr5 and

Lgr4 (pcDNA3.1 backbone and CMV promoter) and ADRA1A-opto] or

available in our laboratory (Airan et al., 2009; Snyder et al., 2013b).

Clones containing bovine GFP–MYO10 (Berg and Cheney, 2002), GFP–

fascin-1 (Adams and Schwartz, 2000) and GFP–VASP (Svitkina et al.,

2003) were gifts of Dr Richard Cheney, and a clone for rat dynamin-1

K44A was available in our laboratory. HEK239 T/17 (HEK) cells were

cultured in 16 DMEM (10-013-CV; Mediatech/Cellgro; Manassas VA)

with 10% FBS (F2442; Sigma; St Louis, MO) and 16 antibiotic-

antimycotic (15240-062; Life Technologies; Carlsbad, CA) according to

the American Type Culture Collection (ATCC; CRL-11268). Cells were

transfected by using calcium phosphate (Kingston et al., 2001) or with

Genecellin (GC1000; BullDog Bio, Portsmouth, NH), where indicated.

Infrared on-cell internalization assayInternalization assays were performed as described previously (Snyder

et al., 2013b) with a few changes. Briefly, Nunc tissue-culture-treated,

clear 96-well plates (167008; Nunc; Rochester NY) were incubated in

50 ml of poly-D-lysine (P0899; Sigma) diluted in H2O to 100 mg/ml for

6 hours at room temperature or overnight at 4 C. Plates were washed with

H2O and air dried in a laminar flow hood. HEK cells were plated at

45,000 cells/well and transfected with the appropriate constructs. For

transfection, calcium phosphate transfection was scaled down to directly

transfect cells in a 96-well format. Briefly, for eight wells of reagent,

2 mg of DNA diluted in 70 ml H2O+10 ml 2.5 M CaCl2 was vortexed,

added to 80 ml 26HBS and aerated, and 20 ml was added per well for 4–

6 hours. The medium was then changed to complete cell culture medium.

To assess the fraction of the receptor on the surface, cells were stained

live with the primary mouse anti-HA antibody (1:500, 50 ml/well) for

45 minutes in clear MEM with supplements, washed once with 150 ml of

clear MEM, fixed (4% PFA) and then stained with goat anti-mouse-IgG

antibody conjugated to Alexa Fluor 680 (Invitrogen, Carlsbad, CA) for

1 hour. Total receptor expression was assessed by first fixing and

permeabilizing cells (0.1% Triton X-100 in PBS for 10 minutes) and then

staining with mouse monoclonal anti-HA for 1 hour in 5% BSA/PBS at

room temperature, followed by detection with a goat anti-mouse-IgG

antibody conjugated to Alexa Fluor 680 for 1 hour. The plate was washed

in PBS for 5 minutes a total of three times and imaged on a LI-COR

Odyssey (Li-COR Biosciences; Lincoln, NE) using the 700-nm channel,

intensity setting of 5.0 and focal offset of 3.0. Untransfected stained cells

were used to subtract background signal from each condition. The

fraction of membrane expression for each receptor was calculated by

taking the ratio of receptor surface expression to total expression. Data

were normalized to the fraction of wild-type membrane expression. The

results of nine independent experiments were averaged.

Live-cell confocal imaging and ultra-long filopodia morphometryA total of 400,000 HEK cells were plated on 35-mm glass-bottomed

dishes (P35G-0-10C; MatTek Corporation; Ashland, MA) that were

coated (glass center only) with 130 ml of 75 mg/ml fibronectin (33016-

015; Life Technologies) for 1 hour in a 37 C incubator. Cells were

calcium-phosphate-transfected with 2 mg of the epitope-tagged receptors,

with or without 2 mg of K44A, and filopodium markers and normalized

to at least 5 mg of DNA with empty vector pcDNA3.1 at 1 day after

plating. The next day, cells were imaged live in clear MEM with

supplements (51200; Life Technologies), 10 mM HEPES (15630; Life

Technologies), 16GlutaMAX-1 (35050; Life Technologies) on a heated

stage set to 37 C on a Zeiss LSM 510 confocal microscope (Carl Zeiss

MicroImaging), using a 1006objective (Zeiss Plan-Apochromat, 1.4 NA,

Zeiss P/N 44 07 80). Based upon our experience in this study and

evidence in the literature (Ramırez-Weber and Kornberg, 1999), long-

filopodia are very fragile and, therefore, live imaging appears to better

preserve their length and number on each cell. However, to assess their

Fig. 7. Lgr5-induced cytonemes provide a platform for engaging barr2.(A) 834del–strawberry (red, left panel) and b2AR–GFP (green, middle panel)were co-transfected and imaged (yellow, colocalization, right panel).(B–D) 834del–strawberry (red, left panel), barr2–GFP (green, middle panel)and HA–b2AR (not shown) were co-transfected and imaged at steady-state(B) or following stimulus of the b2-AR with 10 mM isoproterenol for (C)2 minutes or (D) 20 minutes. Yellow depicts colocalization of the barr2–GFPwith the 834del–strawberry receptor (merge, right panel). Scale bars: 10 mm.

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structural components and determine whether these structures were

present in non-transfected and therefore non-GFP-expressing cells, we

fixed the cells in 4% PFA and performed F-actin staining with Alexa-

Fluor-594-conjugated phalloidin, according to the supplier’s protocol

(A12381, Life Technologies). Briefly, cells were permeabilized with

0.2% Triton X-100 for 20 minutes and then stained with Alexa-Fluor-

594-conjugated phalloidin (1:50) in 0.5% BSA/PBS for 45 minutes at

room temperature, followed by two washes with PBS. This procedure

slightly decreases the size and number of cytonemes, as many cells can

be found that have had their long filopodia severed, especially towards

the distal tips. When described in the text, HA-tagged receptors were

stained as above for on-cell internalization studies but detected with a

goat anti-mouse-IgG secondary antibody conjugated to Alexa Fluor 568

(A11004; Life Technologies). Optical sections of each cell type were

acquired on the confocal microscope and rendered in three dimensions

using the Zeiss LSM Image Browser v4.2 (Carl Zeiss MicroImaging;

Jena, Germany). The number of actin-rich filopodia and their length were

then calculated for each cell by manually tracing and counting each actin-

positive protrusion in the same software package. We analyzed many

cells per genotype using this procedure to provide a large sample size for

statistical measurements as follows: N516 non-transfected; N515

B2AR; N521 Lgr5FL; N514 K44A; N514 Lgr5–V2Rtail; N517

834del; N513 Lgr4.

Immunoprecipitation and western blotting100-mm dishes were plated with 36106 cells, and the cells were calcium

phosphate transfected with 2 mg of each construct as indicated in the

text. Cells were stimulated with 10 mM isoproterenol for the indicated

times and then washed with PBS and scraped in lysis buffer [50 mM

Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, cOMPLETE

EDTA-free protease inhibitors (Roche, Penzberg, Germany) and Halt

Phosphatase inhibitor (78420; ThermoFisher Scientific, Waltham,

MA)]. Lysates were rotated at 4 C for 30 minutes, cleared by

centrifugation at ,13,000 g for 10 minutes, and quantified using a

Pierce bicinchoninic acid assay (BCA) (23225, ThermoFisher

Scientific). 2.7 mg of protein per sample was rotated overnight at 4 C

with 20 ml of anti-FLAG (M2) beads (A2220; Sigma) that were

prepared according to the manufacturer’s suggestion. The next day,

beads were washed (50 mM Tris, 150 mM NaCl, protease inhibitors)

and rotated at 4 C for 5 minutes and then centrifuged at 13,000 g for

1 minute. A total of five washes were performed. Samples were eluted

in 100 ml of Laemmli sample buffer and 5% b-mercaptoethanol for

10 minutes at 55 C, loaded onto 10% Tris-glycine gels (EC6075BOX;

Life Technologies) electrophoresed and transferred to 0.2 mm

nitrocellulose (LC2000; Life Technologies). Blots were Ponceau

stained, cut, blocked in 5% milk/TBST for 30 minutes, washed and

incubated with primary antibodies diluted in 5% BSA/TBST overnight

at 4 C. The primary and secondary antibodies utilized were: rabbit anti-

GFP (ab6556; Abcam; Cambridge, MA) with goat anti-rabbit-IgG

conjugated to Alexa Fluor 800 (611-132-122; Rockland; Limerick, PA);

mouse anti-FLAG (F1804-200ug; Sigma) with goat anti-mouse-IgG

(light-chain specific) (115-005-174; Jackson ImmunoResearch; West

Grove, PA) and donkey anti-goat-IgG conjugated to Alexa Fluor 680

(A21084; Life Technologies); and mouse anti-GAPDH (MAB374;

Millipore; Billerica, MA) with goat anti-mouse-IgG conjugated to Alexa

Fluor 680 (A21058; Life Technologies). Blots were incubated with

the above secondary antibodies, washed and developed on a LI-COR

Odyssey (LI-COR Biosciences).

Fig. 8. Myosin X interacts with barr2.(A) GST–barr2 pulldown of HEK cellsexpressing MYO10–GFP demonstratesthat myosin X interacts with barr2. Theupper panel shows Coomassie staining;the lower panel shows immunoblotting (IB)for GFP. (B) GST–barr2 pulldown frommouse brain lysates identifiesendogenous myosin X as a barr2-interacting protein. The upper panelshows Coomassie staining; the lowerpanel shows immunoblotting. As apositive and negative control for the barr2interaction, b-adaptin and actin wereprobed for, respectively. (C) Cells weretransiently co-transfected with 834del–Lgr5 along with the constructs indicatedand were stimulated with isoproterenol.The FLAG epitope wasimmunoprecipitated (IP) to pull down b-arr2–FLAG, and the blots were probedwith GFP-, FLAG- or GAPDH-specific antibodies.

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barr2–GST pulldownsGST pulldowns were performed according to a previously published

protocol (Marion et al., 2007). Briefly, three whole adult mouse brains or

seven 150-mm plates of HEK cells transiently expressing MYO10–GFP

were extracted in lysis buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA,

1% Triton X-100 and protease and phosphatase inhibitors). Extracts

were incubated overnight with GST or GST–barr2 immobilized on

glutathione–Sepharose high-performance beads (17-5279-01; GE

Biosciences; Pittsburgh, PA). Samples were washed four times, eluted

with reduced glutathione, concentrated using a Ultracel-10K centrifugal

filters (Millipore) and reduced with Laemmli sample buffer. Western

blots were probed with rabbit anti-MYO10 (HPA024223; Sigma), mouse

anti-b-adaptin (610281; BD Biosciences; San Jose, CA) and mouse anti-

actin (MAB1501R; Millipore).

StatisticsData were collected in Microsoft Excel and then transferred to GraphPad

Prism (GraphPad Software; San Diego, CA). Statistically significant

differences between multiple groups were calculated using a one-way

ANOVA and post-hoc Bonferroni.

AcknowledgementsThe authors are thankful for Dr. Scott Soderling’s helpful advice in the preparationand editing of this manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsJ.C.S., L.S.B. and M.G.C. wrote the manuscript. J.C.S., L.K.R. and S.M.performed experiments. J.C.S., H.K.L., L.S.B. and M.G.C. designed experimentsand interpreted experimental results.

FundingThis work was supported by the Susan G. Komen foundation [grant numberKG080627 to J.C.S. and H.K.L.]; and the National Cancer Institute (NCI) ClinicalOncology Research Career Development Program [grant number NCI 5K12-CA100639-10 to J.C.S.]; as well as grants from Duke Cancer Center StewartTrust and Duke Cancer Center Cancer and the Environment to M.G.C.; the NCI[grant number IMAT R21CA173245 to H.K.L., L.S.B. and M.G.C.]; and theNational Institute on Drug Abuse [grant number P30 5P30DA029925-05 to L.S.B.and M.G.C.]. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.166322/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1230–1240 doi:10.1242/jcs.166322

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