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Epidermal keratinocyte polarity and motility requireCa2+ influx through TRPV1

David M. Graham1, Ling Huang3, Kenneth R. Robinson3 and Mark A. Messerli1,2,*1Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543, USA2Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543, USA3Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

*Author for correspondence (mmesserli@mbl.edu)

Accepted 11 July 2013Journal of Cell Science 126, 4602–4613� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.122192

SummaryCa2+ has long been known to play an important role in cellular polarity and guidance. We studied the role of Ca2+ signaling during

random and directed cell migration to better understand whether Ca2+ directs cell motility from the leading edge and which ion channelsare involved in this function by using primary zebrafish keratinocytes. Rapid line-scan and time-lapse imaging of intracellular Ca2+

(Ca2+i) during migration and automated image alignment enabled us to characterize and map the spatiotemporal changes in Ca2+

i. We

show that asymmetric distributions of lamellipodial Ca2+ sparks are encoded in frequency, not amplitude, and that they correlate withcellular rotation during migration. Directed migration during galvanotaxis increases the frequency of Ca2+ sparks over the entirelamellipod; however, these events do not give rise to asymmetric Ca2+

i signals that correlate with turning. We demonstrate that Ca2+-

permeable channels within these cells are mechanically activated and include several transient receptor potential family members,including TRPV1. Last, we demonstrate that cell motility and Ca2+

i activity are affected by pharmacological agents that target TRPV1,indicating a novel role for this channel during cell migration.

Key words: Ca2+, Polarity, Motility, TRPV1, Skin

IntroductionThe building and rebuilding of tissues and organs requires

organization that begins at the single-cell level. Mechanisms that

generate polarity at the single-cell level are common to polarity

at the multicellular level (Nelson, 2003) and, therefore, help

develop a foundation for engineering replacement tissues and

organs. Whereas cytoskeletal elements provide morphological

structure and polarity, a number of upstream signaling cues

provide the information required to organize the cytoskeleton

(Petrie et al., 2009). Ca2+ is known to play a role in directing cell

polarity and guidance in highly polarized processes like tip

growth (Messerli and Robinson, 2007), neurite extension (Zheng

and Poo, 2007), and cell migration (Maroto and Hamill, 2007).

Asymmetries in the distribution of the intracellular Ca2+

concentration [Ca2+]i provide spatial and temporal information,

resulting in directed control of cellular extension and migration.

Ca2+ influx in each of these processes has been linked to

localized activation of mechanosensitive ion channels. In

multiple cases, mechanically sensitive channels provide an

additional degree of cellular polarization through functioning as

chemical receptors, guiding neurite turning (Li et al., 2005; Wang

and Poo, 2005) and lamellipodial extension (Wei et al., 2009).

During tip growth and neurite extension, standing gradients of

[Ca2+]i together with Ca2+ transients at the leading edge, are used

to encode directional information (Messerli and Robinson, 2007;

Zheng and Poo, 2007).

During cell migration, however, Ca2+ signals appear to play

roles at both the trailing and leading edges. Cells extend the

leading edge through protrusion of the lamellipodia in the

direction of migration while simultaneously retracting the

lagging edge (Lauffenburger and Horwitz, 1996). Relatively

steady Ca2+ influx leads to cellular retraction at both the front

(Tsai and Meyer, 2012) and the rear where it enables cells to lift

off the substrate in order to move forward (Lee et al., 1999;

Maroto and Hamill, 2007). Transient Ca2+ influx at the leading

edge of migrating cells promotes lamellipodial extension during

chemotaxis (Wei et al., 2009). Despite the close association of

Ca2+ with these processes, cell migration still occurs in the

absence of extracellular Ca2+ when an electrical polarizing signal

is applied (Brown and Loew, 1994; Fang et al., 1998; Huang

et al., 2009). These seemingly conflicting reports encourage

further investigations into the importance of Ca2+ signals during

migration and in the presence of external polarizing cues.

Therapeutic treatments that impose polarity on cells or direct

outgrowth and migration would be of great significance in

repairing highly organized tissues and organs. Applied electric

fields have shown promise in this regard by directing neurite

extension and cell migration in vitro (McCaig et al., 2005; Zhao,

2009). In the clinic, electric fields have been used in phase I

clinical trials to promote spinal repair (Shapiro et al., 2005) and

have been used successfully for decades in promoting the healing

of chronic wounds (Gardner et al., 1999). However, the

mechanisms by which cells sense and become polarized by

electric fields remain elusive. It has been proposed that electric

fields polarize cells by inducing Ca2+i gradients (Mycielska and

Djamgoz, 2004; McCaig et al., 2005; Zhao, 2009). During

galvanotaxis and galvanotropism, polarized responses are

reduced by inhibitors of Ca2+ influx (McCaig et al., 2005);

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however, they remain unaffected when Ca2+ is removed from theculture medium or when Ca2+

i is buffered by the intracellularCa2+ chelator BAPTA (Brown and Loew, 1994; Fang et al., 1998;Palmer et al., 2000; Huang et al., 2009). Interestingly, weak DC

electric fields and low-frequency AC electric fields (1 Hz)increase Ca2+

i, in vitro (Cho et al., 1999; Huang et al., 2009;Dube et al., 2012). Careful analysis of Ca2+

i changes during

galvanotaxis are necessary to resolve these controversies and mayprovide insight toward the molecular and physical mechanismsby which cells sense and respond to weak DC electric fields.

ResultsCa2+-dependent Polarity and Migration

Cellular migration has long been understood to be dependent onCa2+ signaling (Maroto and Hamill, 2007). We confirmed thisassociation in primary zebrafish keratinocytes by exploring theeffects of Ca2+ availability on cell polarity and motility. As

migratory speed is a function of cellular locomotion speed anddirectional persistence (Lauffenburger and Horwitz, 1996), wehave reported on both parameters. Cells in fish Ringer’s solution

that contained 1.8 mM Ca2+ displayed a bilaterally symmetricmorphology with an average migration speed of 7.560.3 mm/minute. After replacement of extracellular Ca2+ with 1.8 mM free

Mg2+ and EGTA (free Ca2+,1 nM) or buffering of cytosolicCa2+ with the membrane-permeable form of the Ca2+ chelator5,59-difluoro BAPTA, migration speed and average net

displacement were significantly diminished (Table 1). In theabsence of extracellular Ca2+, cells became radially symmetric,giving rise to cell bodies encircled by the lamellipodium. Cellscontinued to migrate – albeit back and forth – over their point of

origin. BAPTA loading of cells approximately doubled thecytosolic Ca2+ buffering capacity (Marks and Maxfield, 1990)and gave rise to cells with irregular morphology, resulting in

tightly adhered fragments or fragments that randomly migrated indirections away from the freely moving cells. These counteringevents often caused cells to undergo severe stretching and sudden

changes in trajectory. Cells treated with 0.5% DMSO behavedsimilarly to cells in normal fish Ringer’s solution (Table 1).These results confirm an important role for trans-plasma-membrane Ca2+ influx in the maintenance of normal cell

polarity and migration.

Lamellipodial Ca2+ sparks

Motivated by the above findings, we mapped the spatial

distribution of Ca2+i within migrating cells (Fig. 1A) and used

an alignment algorithm to enable superimposition of the

individual images to enable further analysis (Fig. 1B,C). Time-lapse imaging of the cytosolic Ca2+ indicator, Indo-1, showed

that the cell body maintains a significantly higher [Ca2+]i than the

lamellipodium (1.360.1 fold, (P,0.001, n55) (Fig. 1D), as is

consistent with previous works (Maroto and Hamill, 2007).Whole-cell Ca2+ pulses were not detected in isolated, migrating

cells, presumably because the long-term absence of serum during

cell sheet dissociation, Indo-1 loading and experimentation.However, small regions of high [Ca2+], termed ‘Ca2+ sparks’,

were identified both within the lamellipodium and along the outer

periphery of the cell body (Fig. 1A). The mean standard

deviation of [Ca2+] changes (Fig. 1E) and the coefficient ofvariation (the ratio of the standard deviation of the pixel

intensities to the mean pixel intensities) (Fig. 1F) indicated

substantially greater variance in the lamellipodia compared withthe cell bodies.

High-speed line-scan imaging was used to characterize thetemporal and spatial nature of the Ca2+ sparks during migration.

Fig. 1G shows an example of consecutive line-scans with time

along the y-axis and Fig. 1H shows the same image after

imposing a threshold of 10% above the mean of the Indo-1 ratiofrom Fig. 1G. Examples of the relative amplitudes of Ca2+ sparks

are shown, including the events 10–50% above the mean Indo-1

ratio (Fig. 1I). The distribution of the duration of Ca2+

sparks (Fig. 1J) was shown to have a mean of 8.765.6

and 10.565.4 milliseconds (n53 for each resolution) for 3-

and 5-millisecond line-scans, respectively (Fig. 1K). For

comparison, cell-attached patch clamp recording was used onmigrating cells to determine a mean open time of

1.8 milliseconds (n55) for inwardly conducting ion channels

on the cell body at resting potential (Fig. 1L). Whereas electricalrecordings enabled capture of 1-millisecond events, both methods

indicate a relatively low abundance of events lasting longer than

a few tens of milliseconds. The Ca2+ spark area in migrating cells

spanned a wide region (Fig. 1M) with mean areas of 2.560.8and 2.261.3 mm2 (Fig. 1N) for scans collected at 3- and 5-

millisecond resolution, respectively.

Lamellipodial Ca2+ sparks were further analyzed to determine

their frequency and distribution during cell migration. Discrete,

spatially localized increases in the Indo-1 ratio increased .10%above the mean of the lamellipodia for, on average, 21.7% of the

time (n528, range 12.6–41.0%). Ca2+ sparks were then analyzed

for their relationship to cellular turning. The results show that the

ratio of the [Ca2+]i magnitude between the left and right halves of

Table 1. Summary of cell motility analyses

Treatment Speed [mm/minute] Distance [mm] Displacement [mm]Persistence

index P-value n Morphology

FR 7.5 (6 2.2) 447.8 (6 132.7) 393.2 (6 134.5) 0.87 - 70 bilateral symmetryEGTA 1.2 (6 0.5) 72.9 (6 30.3) 7.9 (6 6.9) 0.11 ,0.0001* 93 radial symmetryBAPTA-AM 3.7 (6 1.3) 221.5 (6 75.8) 86.1 (6 62.9) 0.39 ,0.0001 88 irregularDMSO 7.7 (6 1.6) 459.7 (6 95.8) 410.3 (6 100.8) 0.89 0.54* 71 bilateral symmetryGd3+ 2.0 (6 0.9) 118.7 (6 51.3) 28.2 (6 21.0) 0.24 ,0.0001* 23 radial symmetryCAP 7.8 (6 1.7) 466.5 (6 102.1) 404.6 (6 114.2) 0.86 0.71 52 bilateral symmetryRTX 6.8 (6 1.1) 407.6 (6 68.8) 358.6 (6 76.0) 0.88 0.0006 54 bilateral symmetryCAPZ 1.8 (6 0.8) 105.8 (6 49.5) 78.8 (6 50.3) 0.73 ,0.0001 76 irregularSB 2.3 (6 0.6) 137.8 (6 38.7) 75.1 (6 42.6) 0.54 ,0.0001 74 irregularRN1734 10.7 (6 2.1) 643.6 (6 126.7) 526.9 (6 156.8) 0.82 ,0.0001 31 bilateral symmetry

*, statistical comparison with FR at top of table. All other treatments were compared with DMSO treatment.RTX, resiniferatoxin; CAPZ, capsazepine; FR, fish Ringer’s; CAP, capsaicin; SB, SB 366791.

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each lamellipodium is not significantly different from 1.0

(P.0.5) for any of the cells, indicating that the amplitude of

the lamellipodial Ca2+ sparks is not associated with cellular

turning (Fig. 2A). A ratio of 1 was determined for both analyses

when the entire area of the lamellipod was used or when only

Ca2+ sparks (10% above the mean lamellipodial Indo-1 ratio)

were analyzed.

Conversely, a significant relationship (P,0.02) was observed

between the relative frequency of the lamellipodial Ca2+ sparks and

rotation rate (Fig. 2B). A greater frequency of Ca2+ sparks on the

right correlated with cells turning clockwise and a greater frequency

of Ca2+ sparks on the left correlated with turning counterclockwise.

These results indicate that frequency and not amplitude of

lamellipodial Ca2+ sparks correlate with cellular turning.

Further analysis of lamellipodial Ca2+ sparks during migration

revealed three separate groups of cells. These cells showed (1) no

turning (n55), (2) turning without a significant difference in the

left:right ratio of Ca2+ sparks (n513) and, (3) turning with a

significant left:right ratio of Ca2+ sparks (n510). Representative

examples of the frequency of Ca2+ sparks and the relative degree

of turning for the different groups are shown in Fig. 2C-N. A

numerical summary of these groups is shown in Table 2. Cells

that migrated without substantial turning did not have significant

differences in the asymmetric frequency of Ca2+ sparks between

the two halves of the lamellipodium (Fig. 2C-E). Turning did

occur in the absence of a significant difference in Ca2+ spark

distribution; however, the direction of turning was random, with

seven cells turning clockwise and six turning counterclockwise

(Fig. 2F-H). When a statistically significant difference in the

left:right Ca2+ spark ratio was measured, cells turned to the side

of the lamellipodium with the greatest frequency of Ca2+ sparks

(Fig. 2I-N). The differences in the left:right ratio of Ca2+ sparks

Fig. 1. Characterization of epidermal keratinocyte lamellipodial Ca2+ sparks. (A) The pseudocolored raw image shows the Indo-1 ratio from a time series

of a migrating keratinocyte. The color bar represents the relative Indo-1 ratio. Scale bar: 10 mm. A greater density of Ca2+ sparks occurs in the lamellipod than the cell

body. (B) Alignment algorithm map of cell area to determine centroid and relative cellular angle about the x-axis. (C) Superimposition of cell outlines after

alignment from time series. (D) Projected mean of Indo-1 intensity values. (E) Projected standard deviation of Indo-1 intensity values for the same cell in panel D.

(F) Illustration of the coefficient of variation of Ca2+ events in the lamellipodium and cell body. The dotted red line indicates the region of the cell from which

the line scan was collected in (G). (G) Representative kymograph of Ca2+ sparks from 5-millisecond line-scans of a migrating cell. y-axis is time, x-axis is distance.

Scale bar is 5 mm. (H) Kymograph of panel G presenting events 10% above the mean of the lamellipodial Indo-1 ratio. (I) Indo-1 ratio intensity plot over time

from panel G (see respective orange and black arrows). Mean intensity5104. 10% incremental threshold values above the mean are identified. (J) Histogram of Ca2+

spark duration from line-scans acquired at 3- and 5-millisecond resolution. (K) Mean duration of Ca2+ sparks. (L) Histogram of mean open time for inward

conducting ion channels. (M) Histogram of Ca2+ spark area from line-scans acquired at 3- and 5-millisecond resolution. (N) Mean area of Ca2+ sparks.

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were consistently statistically significant, even when a more

stringent threshold was used, i.e. 40% above the mean Indo-1 ratio.

In Table 2, directionality of turning cells is presented as clockwise

(right) or counterclockwise (left) for both groups (with and without

a difference in Ca2+ spark frequency) to demonstrate that no

significant difference in Ca2+ sparks existed for either direction

during random turning. Time-accumulated Ca2+ sparks reveal

spatial Ca2+ gradients in motile cells that correlate with turning.

Fig. 2. The frequency of lamellipodial Ca2+ sparks correlates with cell turning. (A) Ratio of the Ca2+ spark amplitude (left:right) of all migratory cells

analyzed, showing no correlation with turning. (B) Ratio of the Ca2+ spark relative frequency (left:right) of all migratory cells analyzed showing a positive

correlation with turning. (C–N) Representative images and corresponding relative rotation plots from populations of cells grouped into four different categories on

the basis of their spatial distribution of Ca2+ sparks and cell turning. Color bar represents the relative frequency of events 10% above the mean lamellipodial Indo-

1 ratio, ranging from 0% (black) to 100% (white). Images and plots demonstrate cells from non-turning (C–E), randomly turning without a significant asymmetric

distribution of Ca2+ sparks (F–H), and left-turning (I–K) and right-turning (L–N) groups with an asymmetric distribution of Ca2+ sparks. Non-turning is defined as

cells with a relative rotation ,2 degrees/minute. Scale bar: 10 mm.

Table 2. Summary of Ca2+ spark frequency and galvanotaxis analyses

Group Ratio of Ca2+ events RatioRotation rate (degrees

per minute)Duration of acquisition

[minutes] n

Straight 1.04 6 0.05 L:R 1.0 6 0.2 7.9 6 1.4 5Left turn with Ca2+ asymmetry *1.40 6 0.14 L:R 9.0 6 1.7 5.4 6 0.9 5Right turn with Ca2+ asymmetry **1.53 6 0.09 R:L -8.7 6 5.5 5.4 6 1.3 5Left turn without Ca2+ asymmetry 0.93 6 0.05 L:R 6.6 6 1.8 4.3 6 0.5 6Right turn without Ca2+ asymmetry 1.09 6 0.06 L:R -8.6 6 0.8 5.3 6 0.5 7EF treatment (cathodal turning) 1.01 6 0.07 cathode:anode 9.3 6 1.9 5.4 6 0.8 10

*, denotes statistically significant from 1. Left turn with Ca2+ asymmetry, *P,0.03; Right turn with Ca2+ symmetry, **P,0.01.

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An applied electric field increases lamellipodial Ca2+

sparks

Previously, we reported that weak DC electric fields increase

[Ca2+]i in keratinocytes but do not generate an asymmetry in Ca2+

magnitude between the two sides of the lamellipodia of cells

(Huang et al., 2009). Here, we tested whether DC electric fields

act by generating an asymmetric distribution of the frequency of

Ca2+ sparks. DC electric fields (100 mV/mm) were applied to

migrating cells while imaging Ca2+i (Fig. 3A). The results

indicate an increase in the occurrence of lamellipodial Ca2+

sparks for 90% of the cell population. In 50% of the cells, this

caused a greater mean [Ca2+]i in the lamellipodia than the cell

bodies (Fig. 3B) and a greater standard deviation (Fig. 3C).

Overall this gave rise to a lamellipodial coefficient of variation of

1.1560.05 (Fig. 3D), which is significantly greater than reported

above in untreated cells (P,0.0001). Despite the greater levels of

lamellipodial [Ca2+], the lamellipodia remained intact, did not

retract and cells continued to migrate (Fig. 3E). Frequency

analysis of the Ca2+ sparks indicates no consistent spatial

increase correlating with cathodal turning (P.0.5). No

statistically significant cathode:anode Ca2+ spark ratio during

turning was found in six of ten cells, and two out of ten cells

showed a significant cathode:anode ratio while turning toward

the cathode and two out of ten cells showed a significant

cathode:anode ratio in the opposite direction of turning. These

data indicate that electric-field-directed migration does notproduce an asymmetric distribution of Ca2+ sparks.

Mechanosensitive Ca2+ influx

Various stimuli for plasma membrane Ca2+ channels were testedto identify the nature of the Ca2+ influx during migration

and galvanotaxis. Gd3+, a blocker of voltage-gated andmechanosensitive ion channels (Yang and Sachs, 1989; Biagiand Enyeart, 1990), significantly reduced single-cell motility

(Fig. 4A), similar to results from cells cultured in Ca2+-freemedium (Table 1). Separation of spontaneous electrical versusmechanical activation was performed by monitoring Ca2+

i using

Fluo-4. Depolarization of the plasma membrane potential with100 mM K+ produced no measureable change in [Ca2+]i (n53cell sheets; data not shown) consistent with an earlier report (Leeet al., 1999). Mechanical activation of cellular Ca2+ channels was

performed by imposing shearing stress through fluid flow acrosssheets of keratinocytes. Prior to flow, transient rises in whole-cell[Ca2+]i occurred for individual cells that remained connected in

cell sheets. Upon application of a transient shearing flow, [Ca2+]i

increased in cells (Fig. 4B) and decayed rapidly in the absence offlow. Further screening for mechanical activation of plasma

membrane ion channels was tested using whole-cell patch clamprecording. Increased current influx occurred when gentle pressurewas applied to the patch electrode (Fig. 4C). Reversal potentialof these currents was near +5 mV. Taken together, these results

demonstrate the presence of plasma membrane, mechanicallygated, non-selective cation channels in epidermal keratinocytes.Plasma membrane nicotinic acetylcholine receptors (nAChRs)

have been identified in mammalian keratinocytes (Kurzen et al.,2007) but do not seem to be present in zebrafish keratinocytes,because neither 100 mM or 1 mM acetylcholine gave rise to

measurable changes in [Ca2+]i (data not shown).

Two gene families of ion channels that have Ca2+ permeabilityand mechanosensitive properties are those of the transient

receptor potential (TRP) and those of the epithelial sodiumchannel [ENaC, also known as sodium channel non-neuronal 1(SCNN1)]. We screened for annotated members of these familiesin zebrafish using the Sanger Institute and GenBank genome

databases. Tissue-specific gene expression analyses wereperformed on the 22 and 6 annotated members of the TRP andENaC gene families, respectively. Gene-specific primers were

generated and optimized using total RNA isolated from adultwhole-body zebrafish samples (Fig. 4D). Several members of theTRP family, including trpa1, trpc6, trpm2, trpm4, trpm5, trpm7,

trpp2, trpv1, trpv4 and trpv6, were consistently detected in adultkeratinocytes. A single ENaC member, asic1.3, was weakly andvariably detected with a reproducibility of two out of ninesamples. These data are shown in the figure as they might be of

importance. The recently identified mechanosensitive channel,Piezo1, was not included in our analysis but is thought to beexpressed in zebrafish skin according to aberrant cell extrusion

function within the epithelium of Piezo1-knockdown animals(Eisenhoffer et al., 2012). The keratinocyte expression profilereflects not only basal keratinocytes (tp63, Bakkers et al., 2002;

Lee and Kimelman, 2002) but the outer epidermal layer (krt8,Martorana et al., 2001), non-keratinocyte epidermal cell lineages(grhl1, Janicke et al., 2010) which include epidermal ionocyte

progenitors, mucus-secreting cells and, perhaps, peripheralneurons (asic1.3, Paukert et al., 2004). Melanophores appear tobe largely absent from the expression profile (c-kit, Kelsh et al.,

Fig. 3. Weak DC electric fields (100 mV/mm) direct migration but do not

generate an asymmetric lateral distribution of Ca2+ sparks.

(A) Illustration of electric field experimental setup. Cells chosen for this study

were migrating perpendicular to field lines prior to the applied electric field.

(B) Projected mean of Indo-1 ratio for a representative cathode-migrating cell.

Color bar represents relative pixel intensity. Scale bar: 10 mm. (C) Projected

standard deviation of Indo-1 ratio for the same cell in panel B. (D) Illustration

of the coefficient of variation of Ca2+ events in the lamellipodium for cells

exposed to the electric field. (E) Relative rotation plot displaying the degree

of rotation of cells over time in an applied electric field. Here, rotation

towards the cathode is defined as ‘positive’ relative rotation.

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2000). The wider expression profile of genes within skin is

probably because of a higher number of other cell types.

TRPV1 is necessary for motility

The role of mechanosensitive TRP channels detected in our

expression analysis was characterized during keratinocyte

migration. TRPM7 has been shown to couple Ca2+ influx to

membrane tension in mammalian fibroblasts (Wei et al., 2009).

The zebrafish tdob508 mutant cell line harbors a channel-inactive

form of TRPM7 (Low et al., 2011) that is lethal early in

development, with fish surviving only into embryonic and post-

embryonic stages (Elizondo et al., 2010). We found that cell

motility and polarity were unhindered in keratinocyte cultures

from the tdob508 mutant line (from 3 days post fertilization, data

not shown). TRPV4 has been shown to be activated by hypotonic

cell swelling and pressure sensation (Suzuki et al., 2003; Vriens

et al., 2004). The TRPV4 antagonist, RN-1734 (Vincent et al.,

2009), had no inhibitory effect on cell migration at

concentrations up to 100 mM (Table 1).

TRPV1 is a well-studied member of the TRP family and is best

known for its activation by vanilloid compounds (e.g. capsaicin,

resiniferatoxin), acid and heat. Mechanosensitive activation has

also been associated with this channel (Birder et al., 2002; Rong

et al., 2004; Jones et al., 2005; Daly et al., 2007). The functional

role of TRPV1 during cell motility was explored by screening

TRPV1-selective agonists and antagonists during cell migration.

No significant change in cell motility was observed upon

treatment of cells with 10 mM capsaicin, when compared to

fish Ringer’s solution or DMSO controls (Fig. 5A,B,E; Table 1).

Treatment of cells with 1 mM resiniferatoxin resulted in a

significant but subtle decrease in cell motility (Fig. 5F; Table 1).

Conversely, treatment with TRPV1 antagonists, 10 mM

capsazepine or 5 mM SB-366791, significantly and reversibly

reduced cell motility, similar to motility of cells that were kept in

Ca2+ free fish Ringer’s solution or were loaded with BAPTA

(Fig. 5C,D,G,H; Table 1). Migration analysis was started

immediately after the addition of any drug in order to remain

consistent with prior analyses even though noticeable effects on

migration did not occur for 5–10 minutes.

In the presence of 10 mM capsaicin, a significant increase in

lamellipodial Ca2+ sparks was monitored, giving rise to a

coefficient of variation that was significantly greater (P,0.02)

than in untreated controls (Fig. 5I,J). Whole-cell Ca2+ pulses were

also stimulated in capsaicin-treated cells, although they occurred in

less than 15% of the total number of images. In the presence of

TRPV1 inhibitors, cells lost their defined lamellipodia, an effect

that prevented Ca2+ spark analysis. As an alternative, we

characterized the changes in whole-cell Ca2+-pulse activity,

which is also due to Ca2+ influx through plasma membrane

mechanosensitive channels (Lee et al., 1999; Huang et al., 2009).

Spontaneous whole-cell Ca2+ pulses from cell sheets increased

,7-fold and 14-fold in response to 10 mM capsaicin (P,0.02) and

1 mM resiniferatoxin (P,0.03), respectively (Fig. 5K). Treatment

with 10 mM capsazepine (P,0.01) and 5 mM SB-366791

(P,0.03) resulted in an ,7-fold and 4-fold decrease, respectively.

During whole-cell patch clamp recording with 1 mM capsaicin

or 30 nM resiniferatoxin in the patch pipette, channel activity in

the plasma membrane increased (Fig. 5L). In the absence of

drugs, single-channel currents were fewer and very brief. The

trace for capsaicin shows increasing activation of plasma

membrane currents as capsaicin diffuses throughout the cell.

Resiniferatoxin commonly caused large cellular currents

immediately after whole-cell clamp was achieved (n57). The

resiniferatoxin trace in Fig. 5L shows progression of ion channel

activity as the drug diffuses through the cell. Capsaicin-activated

currents were outwardly rectified (216.9 pA at 260 mV,

Fig. 4. Ca2+ influx occurs predominantly through mechanosensitive channels. (A) xy-trajectory plot of cells exposed to 10 mM Gd3+ for 1 hour. Scale

bar: 200 mm. (B) Graph from transient shear stress applied to keratinocyte sheets indicates a transient increase in whole-cell [Ca2+]i during application of fluid

flow (n53 cell sheets). (C) Whole-cell voltage clamp recording of a migrating keratinocyte in fish Ringer’s solution. Three times gentle pressure was

applied to the patch pipette, increasing the probability of mechanosensitive channels to be in the open state. Spontaneously activated channels are open briefly.

(D) Tissue-specific gene expression analysis from adult zebrafish keratinocytes of two ion channel families containing mechanosensitive Ca2+-permeable

channels. Genes found at multiple loci were amplified at consensus regions, with the exception of trpc4 and trpc5 that were analyzed separately. The chromosome

number is given for genes that were found at multiple loci. Loading control is b-actin.

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+169.0 pA at +60 mV, n53) – a common feature of TRPV1

channels. Taken together, chemical treatment of epithelial

keratinocytes with known TRPV1 agonists and antagonists

modified Ca2+i in a manner that is consistent with interaction

of TRPV1; however, only the block of TRPV1 channels

significantly reduced migration.

TRPV1 is also activated by acidic conditions. No change of

[Ca2+]i was observed in cells treated by gently adding fish

Ringer’s solution pH 7.0, removing possible activation

contributed by shear stress (Fig. 5M). Exposure to fish Ringer’s

solution pH 5.0 caused a robust yet transient increase in whole-

cell [Ca2+] in all cells within the population. This acid-induced

Ca2+ rise decayed within ,2 minutes (n55). In addition, only

transient Ca2+i increases occurred when medium at pH 7.0 was

completely replaced with medium at pH 5.0 by using a flow

system (n53, data not shown).

In zebrafish, TRPV1 has only been reported in sensory neurons

during embryogenesis (Caron et al., 2008). According to multiple

sequence alignments of the region spanning transmembrane

regions 2–4, which contain residues that are critical for capsaicin

sensitivity, zebrafish TRPV1 is more closely related to chicken

and rabbit than to rat or human TRPV1 and may, therefore,

possess less sensitivity to agonists (Fig. 6A). We did not observe

behavioral responses in animals that were exposed to either

capsaicin or resiniferatoxin starting at 24, 48 or 72 hours post

fertilization (hpf). We report expression of trpv1 early in

development, at the 4–8 cell stage, with increased expression

during morphogenesis of the neural tube (14-somite) (Fig. 6B).

Expression was detected within sensory neurons and the

hindbrain (Fig. 6C). Lower expression was observed in the

embryonic epithelium in animals 24 hours post fertilization

(Fig. 6C arrows). In adult zebrafish, we report widespread tissue

expression of trpv1 that differed from other TRPV members

trpv4 and trpv6 (Fig. 6D). Furthermore, trpv1 expression was

detected in the motile fraction of the adult epidermal epithelia

from keratinocyte explants (Fig. 6E,F). These data indicate that

trpv1 expression is not limited to sensory neurons but is also in

the epidermal epithelium.

Fig. 5. Pharmacological treatment of keratinocytes with TRPV1 agonists and antagonists affect cell motility and Ca2+ dynamics. (A–H) xy-trajectory

plots displaying the migratory paths of individual cells over 1 hour in (A) fish Ringer’s solution (FR), (B) 0.5% DMSO, (C) 5 mM EGTA ([Ca2+]e ,1 nM),

(D) 100 mM BAPTA-AM, (E) 10 mM capsaicin (CAP), (F) 1 mM resiniferatoxin (RTX), (G) 10 mM capsazepine (CAPZ), and (H) 5 mM SB-366791 (SB).

Scale bar: 200 mm. (I,J) Illustrations of the coefficient of variation of Ca2+ events in the lamellipodium of cells in FR and 10 mM capsaicin (CAP). (K) Graph

showing the fold-change in whole-cell Ca2+ bursts from cells exposed to 10 mM capsaicin, 1 mM resiniferatoxin, 10 mM capsazepine, 5 mM SB-366791 and

0.5% DMSO. (L) Whole-cell patch clamp traces at 260 mV holding potential on single migrating keratinocytes exposed to 1 mM capsaicin and 30 nM

resiniferatoxin. Agonists were loaded into the patch pipette and whole-cell recordings were taken immediately after ramp tests. (M) Graphs showing the

population of cells with whole-cell Ca2+ increases upon addition of FR at pH 7.0 (top) and pH 5.0 (bottom) in percent.

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DiscussionDuring cell migration, variations in Ca2+ signals over space, time

and magnitude have made their role difficult to understand. We

chose to study Ca2+ signaling by using one of the most rapidly

migrating vertebrate cell types (zebrafish keratinocytes), which –

at ambient temperature – maintain migratory speeds that are 5–10

times faster than those of mammalian keratinocytes at 37 C

(Maiuri et al., 2012). Zebrafish keratinocytes maintain a high

locomotion speed and a relatively persistent migratory direction

in the presence of normal levels of extracellular [Ca2+], i.e. 1–2

mM. We demonstrate that both speed and persistence are Ca2+-

dependent processes. Disruption of Ca2+i availability by either

blocking Ca2+ influx or buffering Ca2+i results in a loss of

bilateral symmetry and a decrease in persistent migration but

does not inhibit lamellipodial extension or the ability of the cell

to migrate. Lamellipodial extension occurs during low

lamellipodial resting [Ca2+]i (Tsai and Meyer, 2012). We

propose that the substantial decrease in net cell motility in the

absence of extracellular Ca2+ is a consequence of the loss of cell

polarity and the constant shifting of migratory direction in the

absence of polarity. In support of this idea, DC electric fields

rescue polarized migration of zebrafish keratinocytes in the

absence of extracellular Ca2+. Under these conditions, cells

maintained an average speed that is only 55% lower than that of

cells in normal [Ca2+] (Huang et al., 2009). This speed is much

higher than the speed reported here when cells migrate in the

absence of both extracellular Ca2+ and a polarizing applied

electric field.

In visualizing the distribution of free Ca2+i with Indo-1, we

demonstrate a higher, relatively steady [Ca2+]i in the cell body

and Ca2+ sparks in the lamellipodium. Higher [Ca2+]i in the cellbody appears to be a conserved characteristic of motile cells(Maroto and Hamill, 2007). Few articles describe the positivesignaling role of Ca2+ in the lamellipod. Ca2+ influx at the

leading edge of macrophages generates cellular organization(Evans and Falke, 2007), whereas Ca2+ flickers promotelamellipod extension during chemotaxis (Wei et al., 2009).

Untreated keratinocytes in the study by Wei et al. showed Ca2+

sparks in the lamellipodium but still maintained a significantlylower average [Ca2+]i than that found in the cell body. The lower

background [Ca2+] in the lamellipod might provide a bettermeans of detecting the brief, spatially restricted Ca2+ sparks.These brief signals might reflect the rapid speed and highlydynamic nature of fish keratinocyte migration and a requirement

to react more quickly to external cues, compared to the slowermigrating human fibroblasts that show Ca2+ flickers that lastmuch longer (Wei et al., 2009). In the absence of extracellular

Ca2+, there is no difference between [Ca2+]i in the lamellipod andthe cell body, and no noticeable variation in cellular Ca2+ signals(Huang et al., 2009).

Keratinocytes migrate relatively straight or with a slowpersistent turning in the absence of an asymmetric distributionof lamellipodial Ca2+ sparks. However, when there was a

statistically significant asymmetry in the frequency of Ca2+

sparks between the two halves of the lamellipod, keratinocytespivoted about the side with the greater number of sparks. Weinterpret the pivoting as a slowing of the migration of the side

with the greater number of Ca2+ sparks. The amplitude of theseevents does not appear to play a causal role in cellular turning.

The varying levels of Ca2+ during cell migration are consistent

with the Ca2+ model initially proposed for growth cone extensionand retraction (Kater et al., 1988). In keeping with this earliermodel and applying it to migrating cells, we think that long-

duration Ca2+ signals, such as those found in the cell body or thelong-term pulses found in the lamellipod (Lee et al., 1999; Tsaiand Meyer, 2012), lead to membrane retraction. However, brief,spatially restricted sparks that give rise to a lower average Ca2+

signal, modulate the cytoskeleton and promote controlled,directed migration, as reported earlier (Wei et al., 2009). Wesuggest that the asymmetry in lamellipodial Ca2+ sparks that

promotes turning is not sufficient to cause retraction but issufficient to slow lamellipodial extension so that more rapidextension of the other half of the cell leads to cellular turning.

Do Ca2+ sparks direct migration or are they simply a result ofmigration? The fact that cells turn in the absence of Ca2+ sparkasymmetries indicates that they are not just a result of turning. Italso indicates that asymmetries in [Ca2+]i are not required for

cellular turning. Work exploring chemotaxis has shown that Ca2+

sparks are required for directing migration (Wei et al., 2009). Wechose to study galvanotaxis, a mechanism for polarizing cells that

is proposed to work through similar pathways as those usedduring chemotaxis (Zhao, 2009). Epidermal keratinocytespolarize in the presence of weak DC electric fields, a process

that for some time has been postulated to be linked toasymmetries in Ca2+

i signaling (Messerli and Graham, 2011).During galvanotaxis, we observed an increase in lamellipodial

[Ca2+]i in 90% of cells, as they polarized and migrated toward thecathode. The widespread increase in [Ca2+] indicates that a non-graded mechanism of Ca2+ influx is induced by the uniform

Fig. 6. Zebrafish TRPV1 is expressed in the epidermal epithelium.

(A) Sequence alignment of TRPV1 proteins from different species comparing

the critical residues for vanilloid activation. Defined transmembrane regions

(TM) are based on the predicted membrane topology of zebrafish TRPV1.

Red arrows indicate residues important for capsaicin sensitivity and are listed

according to the residue positions of rat TRPV1. (B) Gene expression of

TRPV members during different stages of development. (C) In situ

hybridization for trpv1 in an embryo 24 hours post fertilization (hpf). Arrows

indicate staining in the epidermal epithelium. Scale bar: 250 mm. (D) Tissue-

specific gene expression of TRPV members. (E,F) In situ hybridization of the

motile fraction of an explant of adult epidermal epithelia for trpv1 using

antisense- and sense RNA probes. Scale bar: 50 mm.

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electric field. Despite the comparisons that have been drawnbetween cellular polarization by chemotaxis and galvanotaxis

with respect to Ca2+i signaling, we conclude that they are

different.

Spontaneous Ca2+i sparks have been linked to spontaneous,

localized increases in [Ca2+]i through L-type Ca2+ channels,

ryanodine receptors and Ca2+-induced Ca2+ release in cardiac andsmooth muscle (Kamishima and Quayle, 2003), throughmechanical activation in skeletal muscle (Weisleder et al.,

2012) and fibroblasts through TRPM7 (Wei et al., 2009).Because no measurable Ca2+ influx occurred duringdepolarizing conditions of the plasma membrane in zebrafish

keratinocytes, Ca2+ influx probably occurs through channels withmechanosensitive and/or ligand-gated properties. We proposethat electro-osmotic flow induced by the DC electric field(McLaughlin and Poo, 1981) is generating a shear stress within

the cellular boundary layer that activates Ca2+-permeablemechanosensitive channels over the entire cell surface. In theabsence of the DC electric field, we think that spontaneous Ca2+

influx is also owing to mechanosensitive channels. Under theseconditions, the Ca2+ sparks provide an indication of themechanical stress within the cell. Therefore, mechanical stress

is not just displayed at the trailing edge of the cell but across thelamellipodium as well. These results support efforts to describecell polarity control through membrane tension in the

lamellipodium (Houk et al., 2012). The localized changes inmembrane tension and Ca2+ influx might control Rac and Rhosignaling (Jin et al., 2005; Evans and Falke, 2007; Tian et al.,2010), giving rise to cytoskeletal and morphological polarity.

Based on the mechanosensitive nature of Ca2+ influx in thesecells, we screened two families of ion channels that are known tocomprise mechanosensitive members. We identified ten TRP

channels in the migratory fraction of epidermal explants. Wefound that inhibition of TRPV1, but not TRPV4 or TRPM7,impaired keratinocyte polarity and migration. TRPV1 expression

has been defined mostly in sensory ganglia (dorsal root,trigeminal and nodose) (Caterina et al., 2000), although it hasalso been detected in epithelial cells of the bladder lumen (Birderet al., 2002), epidermal keratinocytes (Denda et al., 2001; Pecze

et al., 2008), as well as other non-neuronal tissue (Southall et al.,2003; Li et al., 2007). We detected robust levels of trpv1 intrigeminal sensory neurons in 24-hour embryos, which is

consistent with its well-documented role in neuronal sensoryfunction. Low levels of trpv1 were detected in the heterogenousembryonic skin; however, trpv1 expression is prominent in adult

epidermal explants. It is important to note that the somas ofsensory neurons have a relatively large diameter, whichundoubtedly influences signal detection through in situ

hybridization. From our gene expression analysis, the fairlywidespread detection of trpv1 in diverse adult tissue types mightreflect a significant role for TRPV1 expression in non-neuronalcell types or might reflect contamination from tissue

innervations, as TRPV1-expressing trigeminal afferents canproject into the innermost epidermal layers (Cavanaugh et al.,2011). By studying the motile fraction of the epithelia that

primarily consist of basal keratinocytes, we avoided suchneuronal contribution to demonstrate trpv1 expression in theepidermal epithelium.

Reversible pharmacological perturbation of keratinocytemigration indicates a novel functional role for TRPV1. Thesignificant and reversible decrease in cell motility and Ca2+

i

activity observed upon treatment with antagonists indicates thatTRPV1-mediated Ca2+ influx is necessary for migration.

Inhibition could not be caused by non-specific inhibition ofvoltage-gated Ca2+ channels or nAChRs, because neithermembrane depolarization (through addition of K+) noracetylcholine treatment gave rise to measureable Ca2+ influx.

Cell motility was not changed in the presence of capsaicin andsubtly decreased in the presence of resiniferatoxin, butlamellipodial Ca2+ sparks and whole-cell Ca2+ bursts

significantly increased. Our finding is in contrast with work inthe slower migrating HepG2 cell line, in which treatment withcapsaicin doubled migration speed (Waning et al., 2007).

Amino acid substitutions in rat TRPV1 have shown thatmutation Y511A resulted in a near-complete loss of sensitivity,whereas mutation S512T resulted in 2-fold decrease in sensitivity(Jordt and Julius, 2002). Furthermore, mutation M547L resulted

in no significant change, whereas T550I resulted in an ,10-foldloss in sensitivity with 1 mM capsaicin; however, 10 mMcapsaicin evoked currents similar to those in wild-type TRPV1

(Gavva et al., 2004). Using the residue positions of wild type andmutant rat TRPV1 for reference, zebrafish TRPV1 containsresidues with no loss (Y511), 2-fold loss (T512), no loss (L547)

and no loss at 10 mM capsaicin (I550). According to primarysequence data alone zebrafish TRPV1 appears capsaicin-sensitive, with reduced sensitivity. It is important to note that

capsaicin sensitivity may also be conferred through othersurrounding residues that have not yet been identified. Thewhole-cell patch clamp recording and the Ca2+-imaging datareported here, show channel activation with capsaicin and

resiniferatoxin. Possible reasons for the lack of behavioralresponses to TRPV1 agonists in zebrafish at 24, 48 or 72 hpfmay include low TRPV1 protein expression during early

development, inefficient permeability through the epithelialmucosal boundary, and/or possible mechanisms that regulateTRPV1 function in different cell types (e.g. covalent

modifications, cofactor binding, splice variants and isoforms).Channel characterization within the endogenous cell environmentand gene expression studies would provide additional insight intothe functional nature of this channel in zebrafish.

Whereas TRPV1 agonists do not appear to be cytotoxic tohuman keratinocytes over brief periods (Pecze et al., 2008),reduced cellular growth and increased apoptosis occur in

epidermal cells of hair follicles during organ culture after a 5-day exposure to 10 mM capsaicin (Bodo et al., 2005). In zebrafishkeratinocytes, TRPV1 agonists induced increases in [Ca2+]i that

are intermittent and not chronic, and that do not result incytotoxic effects for up to 2 hours of exposure. Followingactivation with capsaicin, TRPV1 is desensitized through Ca2+

influx (Caterina et al., 1997), which might prevent further Ca2+

influx in these cells. In neurons, however, where TRPV1 agonistscan be cytotoxic, additional activation of voltage-gated Ca2+

channels through TRPV1-mediated plasma membrane

depolarization may exacerbate Ca2+ influx, leading to toxicity.Pharmacological inhibition of L-type Ca2+ channels attenuatescapsaicin-induced neuronal toxicity (Shirakawa et al., 2008). The

absence of voltage-gated Ca2+ channels in fish keratinocytesmight be important for their survival against TRPV1-mediatedtoxicity.

Last, the mechanosensitive nature of TRPV1 was originallycast in doubt when the TRPV12/2 mice displayed an avoidancebehavior in response to pricking of the hind paw (Caterina et al.,

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2000). Subsequent studies have shown that TRPV12/2 mice showaltered mechanical hypersensitivity in colonic afferents (Joneset al., 2005; De Schepper et al., 2008), bladder afferents (Daly

et al., 2007), urothelial cells (Birder et al., 2001; Birder et al.,2002), osmosensory neurons (Ciura et al., 2011) and muscle (Roet al., 2009). Mechanical activation of TRPV1, through either

direct or indirect mechanisms, might be involved in the mechanicsof Ca2+ influx during the migration of epidermal keratinocytes.

Materials and MethodsKeratinocyte explant culturesScales from an anesthetized adult wild-type zebrafish (Danio rerio) were removedand plated as described previously (Huang et al., 2009). Cell sheets containingprimarily migratory keratinocytes migrated off the scale overnight at 28 C. Cellsheets were dissociated into single cells upon treatment with Ca2+-free fishRinger’s solution (5 mM HEPES pH 7.0, 116 mM NaCl, 2.9 mM KCl, 2 mMMgCl2 and 5 mM EGTA) for 10 minutes at ambient temperature. Scales weremanually removed from the dish after treatment and cells were allowed to recoverin normal fish Ringer’s solution (5 mM HEPES pH 7.0, 116 mM NaCl, 2.9 mMKCl, 1.8 mM CaCl2, 10 mM glucose) for 10 minutes. For single-cell motilityexperiments, cells were further dissociated with a 2-minute treatment in EDTAcontaining fish Ringer’s solution and were incubated in normal fish Ringer’ssolution.

Embryonic keratinocytes from the tdob508-mutant line were obtained fromanesthetized fish. Embryos were dissociated with mechanical perturbation in Ca2+-free medium to obtain single epidermal cells. All animal experiments wereperformed according to approved guidelines.

Cell motility analysisTime-lapse images of migrating keratinocytes were collected from keratinocyteexplant cultures under specified conditions for 1 hour at ambient temperature.Average speed was calculated as the excursion of the cell body over each 30-second time point for each individual cell. Average distance was calculated as thesum of the squares of the differences between two consecutive xy coordinates foreach time point. Single cells chosen for analysis did not contact other cells ordebris during the entirety of the experiment.

The persistence index was calculated as the ratio of the average netdisplacement divided by the product of the mean speed and the time of theexperiment. The range of the persistence index is zero (no persistence) to one(migrating along a straight line away from its point of origin).

Manipulation of Ca2+

Treatments modulating cytosolic Ca2+ were performed with Ca2+-free fishRinger’s solution (extracellular Ca2+ buffering) and normal fish Ringer’ssolution containing 100 mM 5,59-difluoro BAPTA-AM (Molecular Probes,Carlsbad, CA, USA) (intracellular Ca2+ buffering). Cells were loaded with 5,59-difluoro BAPTA-AM for 1 hour at ambient temperature in normal fish Ringer’ssolution containing 0.5% BSA and were gently washed three times in normal fishRinger’s solution prior to imaging.

Ca2+ imagingChanges in [Ca2+]i were measured using fluorescent Ca2+ indicators Indo-1 AMand Fluo-4 AM and (Molecular Probes, Carlsbad, CA, USA). Cells were incubatedin fish Ringer’s solution containing 0.1% BSA (Sigma-Aldrich), 0.001% pluronicF-127 (Molecular Probes), and either 10 mM Indo-1 AM or 5 mM Fluo-4 AM for1 hour at ambient temperature in the dark. After incubation, cells were carefullywashed three times with normal fish Ringer’s solution, left to stand for 30 minutesand gently rinsed again before imaging.

Indo-1 microscopy was performed using two-photon excitation on Zeiss LSMplatforms. Indo-1 was excited with a Coherent Chameleon II laser at 705 or 720 nmand the emitted light was collected with either internal detectors through bandpassfilters 390–465 nm and 500–550 nm or GAsP detectors through bandpass filters414/46 nm and 510/84 nm with a 458 nm dichroic mirror. Fluo-4 was excited with488 nm and emitted light was collected through a 525/45 nm filter with an internaldetector. Rapid line-scans were acquired at 3- and 5-millisecond temporal resolutionwhereas time-lapse images of Ca2+ during migration were collected every 5 seconds.Four consecutive line-scans were averaged for both types of image to reduce noisesuch that each line scan took ,3 milliseconds to acquire. 1286128 pixel time-lapseimages took less than 0.5 seconds to acquire.

Image processingIndo-1 images were lowpass filtered, a ratio of the images was determined, (shortwavelength:long wavelength), masked by the long Indo-1 wavelength andmultiplied by 255 in ImageJ (Rasband, 1997–2012). Binary images of the Indo-1 ratio stack were used to calculate the cellular centroid and the relative angle of its

long axis by calculating the moment about the x-axis using Matlab (Mathworks,

Natick, MA, USA). These values were used to align and rotate images so that their

long axis was parallel to the x-axis.

The left:right pixel intensity ratio (left half:right half) from each half of a cell

was determined for each image in an image stack and the average ratio for theentire image stack was plotted as a function of cellular rotation. The relatively high

resting ratio of Indo-1 in the cell body enabled identification and removal from

analysis.

Image stacks were resampled using Matlab to identify the distribution of pixels

that are more than 10% above threshold. The 10% value was chosen based on

earlier reports of the Indo-1 ratio in cells and the relative intensity changes

measured by other fluorescent Ca2+ indicators in migrating fish keratinocytes(Baker et al., 1994; Lee et al., 1999; Doyle et al., 2004; Huang et al., 2009). This

value provided an objective criterion for analysis and enabled good separation

between the background variation of the lamellipodial intensity and the Ca2+

sparks. Threshold was applied to each image and pixels above threshold were

counted. The resultant image for each image stack reflects the spatial distributionof the number of times that the relative Indo-1 ratio was above threshold. The

left:right pixel intensity ratio of the two halves of the cell enabled comparison of

relative frequency of Ca2+ sparks. The ratios were plotted against cellular rotation

where negative rotation corresponds to counterclockwise rotation and the positiverotation corresponds to clockwise rotation.

Electrophysiology

Whole-cell and cell-attached patch voltage recordings were performed on isolated,

migratory keratinocytes in fish Ringer’s solution at ambient temperature. Cells

were dissociated with Ca2+-free fish Ringer’s solution as described above and

treated for 30 seconds with 0.05% Trypsin-EDTA (Gibco, Carlsbad, CA, USA) toimprove high-resistance seals. Micropipettes (VWR Scientific, West Chester, PA)

were pulled and fire polished to give tip resistances of 2–3 MV. Backfilling

solution for whole-cell recordings consisted of (10 mM HEPES pH 7.2,150 mM

KCl, 2 mM MgCl2, 5 mM EGTA), whereas cell-attached patch electrodes were

backfilled with fish Ringer’s solution. Voltage clamp recording was performedwith an Axon Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA,

USA) running with pClamp 9.2 software. Current records were acquired at 20 kHz

with the lowpass input filter set at 5 kHz. TRPV1 agonists, capsaicin and

resiniferatoxin, were mixed in with the backfilling solutions prior to obtaining.3 GV seals. In whole-cell clamp recording capsaicin was maintained at 1 mM

and resiniferatoxin ranged from 30–300 nM. During cell-attached patch, final

concentrations of 10 mM capsaicin and 1 mM resiniferatoxin were used in the

patch pipette.

Gene expression analysis

Total RNA was isolated from either whole body, skin (devoid of scales and

dissected from the lateral side of the fish) or keratinocyte explant cultures. Whole-body and tissue-specific RNA was isolated from adult wild-type fish essentially as

described (Westerfield, 2000). All tissue and/or cell lysates were extracted in TRI

reagent solution (Ambion, Carlsbad, CA, USA) as described by manufacturer.

Total isolated RNA was treated with DNase I (New England Biolabs, Ipswich,MA, USA) for 30 minutes at 37 C and purified with the RNeasy MinElute Cleanup

Kit (Qiagen, Valencia, CA, USA). RNA was converted to complementary DNA by

using RT-PCR with M-MuLV reverse transcriptase (NEB) and subsequent PCR

was performed on designated targets. A full list of primers can be found insupplementary material Table S1.

Pharmacology

Pharmacological treatment with TRPV1 antagonists and agonists for cell motilityand Ca2+ imaging were performed with 10 mM capsazepine (Tocris bioscience,

Bristol, UK), 5 mM SB-366791 (Tocris bioscience), 10 mM synthetic capsaicin

(N-vanillylnonanamide; Sigma-Aldrich, St Louis, MO, USA), and 1 mM

resiniferatoxin (Tocris bioscience). Control treatment used was 0.5% (v/v)DMSO. Treatment of 24–72 hours zebrafish was performed with 100 mM

capsaicin and 10 mM resiniferatoxin in E3 medium (1 mM HEPES pH 7.4,

5 mM NaCl, 0.17 KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4) for ,1 hour at

ambient temperature.

Whole-mount and explant in situ hybridization

The trpv1 RNA probe was prepared from plasmid pCDNA3-TRPV1 by PCR

amplification of the full-length TRPV1 encoding region (2460 bp) and in-vitrotranscription with SP6 RNA polymerase (NEB). The RNA probe against trpv1 was

labeled with DIG 11-UTP (Roche, Indianapolis, IN, USA) and detected with an

anti-DIG antibody (alkaline phosphatase Fab fragment) (Roche) using NBT/BCIP

(Roche), as described previously (Thisse et al., 2004). Both wild-type developingembryos and adult keratinocyte explant cultures were fixed with 4%

paraformaldehyde in phosphate-buffered saline and processed as previously

described (Thisse et al., 2004).

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Statistics

Student’s t-test was used to determine statistical significance for the comparisonbetween two groups. Regression analysis was used to determine significance ofcellular rotation versus Ca2+ spark amplitude and versus Ca2+ spark frequency.

AcknowledgementsWe thank Steve Correia and Nikohl Graham for their assistance withstatistics, Alexander Schier’s lab for providing the pCDNA3-TRPV1construct, Rob Cornell and Greg Bonde for the tdob508 mutant line,Casey Kraft and Douglas Richardson for their help with advancedimaging instrumentation, and Robert Prendergast for informativediscussions.

Author contributionsD.M.G., L.H., K.R.R. and M.A.M. designed experiments; D.M.G.,L.H. and M.A.M. performed experiments; D.M.G. and M.A.M.analyzed data; and D.M.G., K.R.R. and M.A.M. wrote the paper.

FundingThis work was funded by NIH P30GM092374 to G.G. Borisy, theEugene and Millicent Bell Fellowship Fund in Tissue Engineeringand the Herman Research Award to M.A.M. Deposited in PMC forrelease after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122192/-/DC1

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