4.1.

Cell Culture

Experiments were carried out with a mouse cell line derived from the cortical collecting duct [mCCD 1296 (d)] of a heterozygous offspring of the Immortomouse (Charles River Laboratories, Wilmington, Massachusetts). The Immortomouse carries as transgene a temperature-sensitive SV40 large T antigen under the control of an interferon- $\gamma$ response element. Cells were maintained on collagen-coated Millicell-CM inserts (Millipore Corp, Billerica, Massachusetts) to promote a polarized epithelial phenotype. Cells were grown to confluence at $33°\mathrm{C}$ , 5% $\mathrm{C}{\mathrm{O}}_2$ and then maintained at $37°\mathrm{C}$ , 5% $\mathrm{C}{\mathrm{O}}_2$ to enhance differentiation. The growth medium consisted of the following (final concentrations): Dulbecco’s Modified Eagle Medium (DMEM) w/o glucose and Ham’s F12 at a 1:1 ratio, $5\mathrm{mM}$ glucose, $5\mu \mathrm{g}∕\mathrm{ml}$ transferrin, $5\mu \mathrm{g}∕\mathrm{ml}$ insulin, $10\mathrm{ng}∕\mathrm{ml}$ epithelial growth factor (EGF), $4\mu \mathrm{g}∕\mathrm{ml}$ dexamethasone, $15\mathrm{mM}$ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.06% $\mathrm{Na}\mathrm{H}\mathrm{C}{\mathrm{O}}_3$ , $2\mathrm{mM}$ L-glutamine, $10\mathrm{ng}∕\mathrm{ml}$ mouse interferon- $\gamma$ , $50\mu \mathrm{M}$ ascorbic acid 2-phosphate, $20\mathrm{nM}$ selenium, 5% fetal bovine serum (FBS). For differentiation, FBS, insulin, and interferon- $\gamma$ were omitted from the apical medium and insulin, EGF, and interferon- $\gamma$ from the basal medium. The cells developed cilia and became fully differentiated (from transepithelial electrophysiology; see Ref. 10) after $2\text{weeks}$ of culture in differentiation conditions.

An en face image of the cultured cells after complete differentiation is shown in Fig. 1 .

Fig. 1

En face view of terminally differentiated mCCD monolayer. Scale bar is 5 microns. Arrows indicate primary cilia, which grow perpendicular to the cell surface. Typically, between 50% to 80% of cultured cells contain a primary cilium. Inset is a magnified and contrast-enhanced section showing the middle cilium.

4.2.

Calcium Fura-2 Protocol

The protocol follows that from Nauli.¹² A stock solution of $1\mathrm{mM}$ Fura-2AM was first prepared, consisting of $1\mathrm{mg}$ Fura-2AM into $1\mathrm{mL}$ dispersant solution, consisting of $20\mathrm{wt}\mathrm{\%}$ Pluronic F-127 in $80\mathrm{wt}\mathrm{\%}$ dimethyl sulfoxide (DMSO). The cells were incubated with the stock solution diluted down to $5\mu \mathrm{M}$ at $37°\mathrm{C}$ for $30\min$ . The monolayer was washed three times with DMEM to remove excess Fura-2AM and the insert placed into a $60\text{-}\mathrm{mm}$ culture dish with DMEM (phenol red indicator omitted, $1\mathrm{mM}$ probenecid added), which was then placed within the microscope incubation chamber. The illuminator source was a Lambda DG-4 switchable source that alternately illuminated the cells with $340\text{-}\mathrm{nm}$ and $380\text{-}\mathrm{nm}$ light, corresponding to the different absorption peaks of Ca⁺⁺-free and Ca⁺⁺-bound states of Fura-2. A dichroic filter cube (Chroma filter set 71000A) within the microscope was used to illuminate and detect the Fura-2.

4.3.

Optical Tweezers

See Fig. 2 . The source was a Crystalaser IRCL- $0.5\text{-}\mathrm{W}$ -1064, a diode-pumped Nd:YAG continuous-wave single-mode laser providing $0.5\text{-}\mathrm{W}$ optical power from a $10\text{-}\mathrm{W}$ electrical power supply. The optical tweezer breadboard layout was constructed using Linos Microbench optomechanical mounts. Achromatic doublets were used for the beam expansion. The first lens has a $10\text{-}\mathrm{mm}$ focal length, while the second has a $200\text{-}\mathrm{mm}$ focal length. Both lenses were antireflection coated for $1064\mathrm{nm}$ . The focal lengths were chosen simply for convenience: the distance between the entrance port of the microscope and the objective lens is $140\mathrm{mm}$ , and the laser beam was expanded a factor of about $6\times$ to fill the aperture. The objective lens used was a Leica $63\times$ NA 0.9 U-V-I HCX long working distance plan apochromat dipping objective with a $2.2\text{-}\mathrm{mm}$ working distance. The tweezer couples into the microscope through an existing lateral port. A side-looking $1064\text{-}\mathrm{nm}$ dichroic mirror (Chroma) mounted within the fluorescence turret provides the ability to perform normal transillumination microscope viewing while the tweezers are operating. A KG-1 IR cutoff filter (Newport) was permanently installed within the imaging path above the fluorescence turret to attenuate the Nd:YAG beam during alignment and trapping operations. The small amount of leakage from the backscattered tweezer light through the two dichroic filters allows observation of the tweezer spot by a camera during alignment and operation. The trapping beam was used to apply a force to individual cells by moving the microscope stage; the trapping beam does not move.

Fig. 2

Laser tweezer apparatus. The laser tweezer is coupled into the fluorescence turret via a side-look mirror and is located outside the incubation chamber.

4.4.

Microscopy

Imaging and manipulations of terminally differentiated epithelial monolayers were carried out using a Leica DM 6000 upright microscope equipped with a heated and $\mathrm{C}{\mathrm{O}}_2$ controlled incubation chamber (Solent Scientific). Calcium imaging was performed using a Princeton Instruments 512 EMCCD camera and a Sutter Instruments Lambda DG-4 switchable source. Bright-field images and optical trap monitoring was performed by a Point Grey Instruments “Flea” camera. Calcium images were acquired using MetaFluor, and typical exposure times were $300\mathrm{ms}$ . Image pairs were acquired every $10\mathrm{s}$ and analyzed using MetaFluor Analyst. Monolayers were found to be viable for up to $4\mathrm{h}$ , “viability” defined here as a Ca⁺⁺ response to adensosine triphosphate (ATP) stimulation, and in addition, maintenance of transepithelial resistance.

4.5.

Microscope Sample Stage

Because of the nature of the experiments presented here, I present some information regarding the sample stage. The motion in $z$ (along the optical axis) was controlled by the Leica microscope, a microstepper motor with a resolution of $0.015\mu \mathrm{m}$ . A display allows quantitative control of the $z$ -stage displacement. Motion in $x$ and $y$ was accomplished by use of a motorized Prior stage (H101A ProScanII) with a minimum step size of $0.01\mu \mathrm{m}$ .

4.6.

Tweezer Characterization

A complete characterization of the trap stiffness is beyond the scope of this paper. Rather, the relevant information is simply that the force applied to a cilium is within a physiologically relevant range. Two methods were used to assess the maximum applied force: holding a sphere and moving the stage,³⁷ and holding a sphere in an applied flow via a flow chamber.³⁶ Polystyrene microspheres suspended in phosphate-buffered saline (PBS) were used. A microsphere was trapped far from a surface, and the sample stage moved at a slowly increasing velocity until the sphere fell out of the trap. The trapping force is then (approximately) given by the Stokes drag force. This method correlated well with the alternative method, holding a sphere stationary in the presence of applied steady flow created by a digitally controlled peristaltic pump (Ismatec REGLO ISM 834). It should be noted that use of long tubes of small diameter connecting the pump to the chamber caused the flow within the flow chamber to be steady rather than rectified. Both measurements indicated the trap strength of a $2\text{-}\mu \mathrm{m}$ -diam latex sphere in PBS of approximately $210\mathrm{pN}$ . This is in the range of forces within a renal tubule,³⁸ indicating that a trapped sphere can be used to apply a physiologically relevant force to a cilium.

4.7.

Calcium Imaging and Trapping

An example ratiometric image is shown in Fig. 3 . With Metafluor running and acquiring images, the filter turret was manually rotated from the Fura-2 cube to the tweezer cube and the illumination changed to bright field.

Fig. 3

Example ratiometric Fura-2 image. This image represents the ratio of two images, one acquired using $340\text{-}\mathrm{nm}$ excitation and the other using $380\text{-}\mathrm{nm}$ excitation. The circled cell is the one that was manipulated with the laser tweezer. Scale bar is 5 microns.

Observing the trapped particle and monolayer with the Flea camera, the trapped particle was rubbed either against the apical surface or against the primary cilium of a single cell several times before returning the microscope back to calcium imaging configuration. A consequence of this procedure is that a few frames during the time course contain artifacts of the Nd:YAG illumination and are consequently omitted from the presented data. It should be noted that the combination of the dichroic mirror and the KG-1 filter did not completely eliminate the backscattered trapping illumination. This is due to the extreme sensitivity of the CCD array.

4.8.

Sample Size

Multiple measurements were performed on multiple monolayers. Each experimental result presented here represents results obtained on four different confluent monolayers (with passage numbers between 42 and 54), each monolayer used up to five times (contingent on monolayer viability), and each daily individual experimental run was performed on three different cells from a single confluent monolayer. This sample size, combined with the clonal nature of the cell line, indicates the results obtained are robust.

5.1.

The Tweezers Can Directly Trap a Cilium with Sufficient Force to Move It

There are two ways the optical tweezers can be used to apply a force to a primary cilium. One way is to trap a small object (e.g., microsphere) and, using the object, push against the cilium. A second way is to directly trap the cilium and move the cell, causing a deformation in the cilium to occur. The advantage of the first method is that the trapping force, and thus the force applied to the cilium, can be calculated much more accurately. The advantage of the second is that the tweezers apply a force to the cilium without any sort of material contact occurring. The disadvantage of the first method is that downstream signaling effects could be caused by the physical contact between trapped object and cilium rather that the applied stress itself. The disadvantage of the second method is that the applied force is known much less accurately, due to lack of detailed knowledge of the optical properties of the cilium, the high aspect ratio (length/diameter $\sim 20$ ) of the cilium, and the fact that a cilium is anchored at one end to the cell. Because use of a trapped particle to deform a primary cilium will allow for the possible confounding effects of physical contact, I attempted to directly trap a primary cilium with the optical tweezers and cause the cilium to deform. Given the small diameter of the cilium $\left(0.2\mu \mathrm{m}\right)$ , it was expected that any trapping force would be small, possibly insufficient to cause ciliary deformation. Example results are shown in Fig. 4 .

Fig. 4

Optically trapped cilium. Scale bar is 5 microns. Arrow indicates the cilium. Frame (d) is a subtraction of frame (c) from frame (a) and clearly indicates that the cilium can be moved directly by the optical trap in spite of the submicron dimensions of the cilium.

These images are taken from a video sequence and show that a primary cilium can be stably trapped and held in the trap with sufficient force to allow for significant deformation. Using published data for the mechanical stiffness of the primary cilium,²⁵ applying the trap to the distal tip of the cilium, and measuring the maximal deflection of the cilium tip from acquired images allowed the deformed cilium to be modeled as a homogeneous cylindrical cantilevered beam³⁸ and thus estimate the applied force. Using this data, the applied force was estimated to be approximately $1\mathrm{pN}$ . Based on perfused tubule data,³⁵ this is not a physiologically relevant force. Consequently, trapped particles were used to apply mechanical disturbances to cilia for the remainder of this study.

5.2.

Trapping Does Not Damage the Monolayer

It is important to note that confluent monolayers represent a functional entity that is highly sensitive to even small changes in individual constituent cells.³⁹ Specifically, loss of, or damage to, a single cell is sufficient to render the entire monolayer nonfunctional. Even though the trapping wavelength was chosen to minimize absorption and photodamage,⁴⁰ because monolayer integrity is so different from apoptosis of isolated cells, it was necessary to determine that the trapping laser did not irreparably damage the monolayer, either directly through photodamage and heating, or indirectly by heating the apical medium. The trapping laser was trained on a single cell and held in place for several minutes, representing a much longer exposure than occurs for trapping and stimulation. The monolayer (still within the culture well) was then placed into a voltohmeter (World Precision Instruments Endohm chamber), which showed that the transepithelial resistivity remained undisturbed [monolayer resistance was typically $1\text{to}2\mathrm{k}\Omega \cdot {\mathrm{cm}}^2$ ; Ref. 10)]. There was evidence that the trapping beam heated the medium, as seen by the appearance of large-scale convective motion, which would bring cellular debris from elsewhere into the field of the view of the camera. However, no flow-induced biological effects were observed (as verified by ${\mathrm{Ca}}^{+}$ ratiometic imaging). The induced flow did not appear to damage the viability of the monolayer either, also verified by transepithelial electrophysiology. Consequently, although the tweezer beam may induce biological effects, the readouts chosen here are not affected.

5.3.

Applying the Tweezer Directly to the Cell Membrane Does Not Elicit a Calcium Response

Two procedures were performed to ascertain whether application of the tweezer beam would result in the release of intracellular ${\mathrm{Ca}}^{++}$ . The trapping beam was applied directly to the apical cell membrane, far from the primary cilium. For one set of experiments, the trap was held steady in one position for a minute. For other experimental runs, the cells were moved in an oscillatory fashion for one minute, keeping the trap far from the primary cilium. The time scale was chosen based on flow-type stimulation, in which the release of intracellular calcium occurs very rapidly upon onset of flow.¹² An example trace of the measured fluorescence ratio is shown in Fig. 5 . The graph shows no significant change in the concentration of cytosolic Ca⁺⁺ mechanotransduction pathways. This demonstrates that the optical trap, in itself, does not cause the release of intracellular ${\mathrm{Ca}}^{++}$ .

Fig. 5

Ratiometric Fura-2 trace of a region containing a cell stimulated by the laser tweezer beam. An apical region of a cell, far from the primary cilium, was illuminated by the trapping laser. Scale bar indicates the time of illumination. Ratiometric image acquisition was paused during tweezer stimulation.

5.4.

Physical Contact of a Trapped Particle Against the Cell Membrane Does Not Elicit a Calcium Response

In another experiment, a trapped particle was pushed into physical contact with the apical surface of a cell (far from a cilium). Because the axial intensity gradient is approximately five times less than the radial gradient for an NA 0.9 beam,⁴¹ the estimated maximum downward force exerted on the membrane was $50\mathrm{pN}$ . The actual force applied was likely substantially lower, corresponding to an observed bead displacement of approximately $1\text{micron}$ , based on $z$ -axis displacement of the microscope stage while maintaining the bead in optical focus. This experiment was performed to more closely emulate the micropipetting technique of Praetorius and Spring² and Sanderson and Dirksen.⁴² The trapped particle was sinusoidally oscillated along the surface for one minute. The results of the Fura-2 images are shown in Fig. 6 .

Fig. 6

Ratiometric Fura-2 trace of a region containing a cell stimulated by a trapped particle. The trace was calculated based on a region of several cells. An apical region of a single cell, far from the primary cilium, was stimulated by a trapped particle for $1\min$ . Scale bar indicates duration of stimulation. Ratiometric image acquisition commenced upon cessation of stimulation.

Interestingly, stimulation of the apical surface by physical contact did not induce the release of calcium into the cytosol, indicating that any mechanotransduction apparatus is not likely located indiscriminately along the apical surface. One important and relevant distinction between this result and the contrary result reported by Sanderson ⁴³ is the magnitude of applied force; Sanderson’s work reported pushing a micropipette $6\mu \mathrm{m}$ into the cell membrane, while the deformation reported here is substantially less.

It should be noted that the null results obtained here and earlier collectively imply that any induced forces caused by motion of the microscope stage are also insufficient to provoke intracellular ${\mathrm{Ca}}^{++}$ release.

5.5.

Pushing a Trapped Microsphere against the Cilium Correlates with the Release of Intracellular Ca⁺⁺

Here, a trapped microsphere was brought into contact with the cilium once, with a typical Fura-2 trace shown in Fig. 7 . Clearly, contact with the primary cilium did elicit a response. Based on the trap characterization data and the size of the trapped particle,³⁷ the applied force was not more than $200\mathrm{pN}$ . The interpretation is that a localized force applied directly to the primary cilium elicited release of intracellular ${\mathrm{Ca}}^{++}$ , in agreement with previous reports.² This result, taken with the lack of intracellular calcium release when a force is applied elsewhere on the apical surface, implies that the primary cilium is the seat of mechanosensation in renal epithelia.

Fig. 7

Ratiometric Fura-2 trace of a region containing a cell stimulated by a trapped particle. Here, a primary cilium was manipulated as Fig. 7, and the Fura-2 trace was calculated over a region of several cells. Scale bar indicates time and duration of stimulation. The small spike is an artifact of the experimental procedure.

5.6.

ATP Applied Apically Did Elicit Intracellular Ca⁺⁺ Release (Not Due to Flow)

As a final control, ATP was acutely applied to the apical media. This was regularly done as a way to check the overall viability of the monolayer, as ATP is a potent agonist of Ca⁺⁺ release. Using a syringe and micropositioner, a small volume (several microliters, representing a sub 0.1% change in apical volume) of ATP in DMEM was added to the apical surface. The final ATP concentration, after addition to the apical surface, was approximately $1\mathrm{mM}$ , well in excess of the binding constant. The Ca⁺⁺ trace (Fig. 8 ) shows the characteristic response due to the presence of P2-purinergic receptors.⁴⁴ It is important to note that this response was not due to flow induced by the addition of agonist; this was verified by adding DMEM alone at the same rate of application (results not shown).

Fig. 8

Ratiometric Fura-2 trace of a region containing cells stimulated by apical addition of $2\mathrm{mM}$ ATP. Scale bar indicates time of addition of solution. The small spike is an artifact of the experimental procedure.

6.1.

Magnitude of Applied Force

The specific deformation state of the cilium (and resultant elastic strain energy) is highly dependent on the distribution of load along the length as well as the mechanical properties of the cilium. While very little detailed experimentation to measure the physical properties of the cilium has been performed,³⁸ preliminary results indicate that the cilium can be adequately modeled as a homogeneous slender cantilevered cylinder with a free end and a built-in end, and with a bending modulus of $31{\mathrm{pN}}^{*}\mu {\mathrm{m}}^2$ (Ref. 25). To simplify interpretation of the experimental results presented here, the force was consistently applied as close to the free end of the cilium as possible. Numerical solution of this system has been performed, and preliminary results have been presented elsewhere.³⁸ Those prior results provided much of the justification for the experimental work presented here.

6.2.

Stimulation of the Apical Surface

There exists a sizable body of evidence that the molecular origin of mechanosensation may not be related to the primary cilium (see, for example, Refs. 32, 45). It is important to note that this evidence is predicated on fluid flow stimulation of epithelia—that is, the applied force was distributed over the entire apical surface. If direct stimulation of the apical membrane can induce a release of intracellular Ca⁺⁺, one interpretation would be that mechanosensation is not strictly mediated by the primary cilium, and that mechanosensitive proteins exist indiscriminately on the apical membrane.³³ However, the experimental results indicate that direct stimulation of the apical surface, with or without physical contact with the membrane, does not elicit an intracellular Ca⁺⁺ release. These results indicate that in principal cells of the cortical collecting duct, the apical membrane does not participate in mechanotransduction.

6.3.

Statistical Significance of Results

The various Fura-2 curves were obtained for multiple experiments, performed on multiple cells within multiple confluent monolayers. Thus, the results presented are robust against cell-to-cell variations as well as variations of the amount of Fura-2 dye loaded into the cell. Pharmacological manipulations allowing quantitative conversion of the dual-wavelength ratio to calcium concentration⁴⁶ were not performed at the time due to effects of calcium deprivation on the integrity of the monolayer.⁴⁷

6.4.

Effects of Applying a Localized Disturbance

The major difference between previous reports and the results reported here is the use of a method that applies a highly localized disturbance—the spatial extent of the applied disturbance is on the order of a square micron or less. Consequently, this experimental method does not suffer form any ambiguity about where the applied forces act. The results presented here, taken together, tell a compelling story—it is possible to apply a localized force directly to a primary cilium using laser tweezers, the magnitude of this force can generally exist within a physiologically relevant range, and application of a localized force directly to a primary cilium elicits a physiological response, as previous reports indicate. This report also presents preliminary data that apical force sensing occurs only via the primary cilium, and that the mechanosensitive apparatus is not indiscriminately located on the apical surface. One possible explanation between the null results reported here and positive results reported by other groups is that the actin-integrin force sensing apparatus occurs on the basolateral surface, which can be stimulated when the entire monolayer is subjected to shear stress on the apical surface.

At this time, the apparatus is not able to directly trap a cilium (or bead) while simultaneously performing Fura-2 ratiometric imaging due to both beamlines sharing the fluorescence turret. Consequently, one remaining potential ambiguity is that the cilium-mediated response was obtained by physical contact of a trapped particle onto the cilium, rather than a mechanosensation mechanism. Even so, the data presented here is sufficient to distinguish between cilum-mediated mechanotransduction and alternative hypotheses.