Control of cell membrane tension by -I

Rajalakshmi Nambiar, Russell E. McConnell, and Matthew J. Tyska1

Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37205

Edited by Edward D. Korn, National Heart, Lung and Blood Institute, Bethesda, MD, and approved May 29, 2009 (received for review February 13, 2009) All cell functions that involve membrane deformation or a change not been explored. Thus, the goal of the current study was to in cell shape (e.g., endocytosis, exocytosis, cell motility, and cyto- determine whether class I function in controlling the kinesis) are regulated by membrane tension. While molecular mechanical properties of the plasma membrane. contacts between the plasma membrane and the underlying are known to make significant contributions to mem- Results brane tension, little is known about the molecules that mediate Probing Membrane Tension with an Optical Trap. We sought to these interactions. We used an optical trap to directly probe the investigate the contribution of Myo1a and other class I myosins molecular determinants of membrane tension in isolated or- to plasma membrane tension in isolated organelles and living ganelles and in living cells. Here, we show that class I myosins, a family of membrane-binding, actin-based motor , mediate cells. To this end, we developed an optical trap assay that enabled membrane/cytoskeleton adhesion and thus, make major contribu- us to measure the force exerted by a thin tubule or ‘‘tether’’ tions to membrane tension. These studies show that class I myosins extracted from a membrane (14). In a typical tether force ␮ directly control the mechanical properties of the cell membrane; experiment, a concanavalin-A-coated 2.0 m diameter micro- they also position these motor proteins as master regulators of sphere was captured in the optical trap and then brought in cellular events involving membrane deformation. contact with an isolated brush border or intact cell, which was firmly attached to a glass coverslip surface. Membrane tethers actin ͉ brush border ͉ cytoskeleton ͉ microvilli ͉ optical trap were then formed by translating the piezoelectric stage to move the sample away from the trapped bead. Forces exerted by lasma membrane tension in eukaryotic cells is a master membrane tethers on the bead were derived from microsphere Pregulator of all cellular processes that involve membrane position data (15), acquired at video rate using a CCD camera; deformation including endocytosis (1), exocytosis (2), mem- position data were converted to force using the stiffness of the brane repair (3), cell motility (4), and cell spreading (5). The optical trap (kTrap), which for our studies ranged from 0.1–0.4 total tension present in the plasma membrane (i.e., the ‘‘appar- pN/nm (Fig. S1). ent’’ membrane tension, TApp) has a minor contribution from the surface tension of the lipid bilayer (TM) and a substantial Myo1a KO Brush Borders Demonstrate Defects in Membrane Force- contribution from the molecular contacts that afford adhesion to Extension. As a first step, we examined the force-extension ␥ the underlying actin cytoskeleton ( ) (6). To prevent large properties of the apical membrane associated with brush borders changes in tension, the plasma membrane must maintain con- isolated from WT or Myo1a KO mouse small intestine. Because tinuous interactions with the cytoskeleton, despite the fact that isolated brush borders are prepared in the absence of ATP, this structure is highly dynamic and continuously remodeling. tether formation in this case is an irreversible process. The first However, little is known about the molecules that mediate step in these experiments was to capture a ConA-coated bead dynamic interactions between membrane and cytoskeleton, or with the optical trap and bring it in contact with a coverslip- the proteins that contribute directly to controlling membrane adsorbed brush border. After waiting approximately4stoallow tension in cells. for bead binding to the apical membrane, tethers were pulled by A striking biological example of the complex mechanical translating samples away from the trapped bead at 1.0 ␮m/s (Fig. interplay between the plasma membrane and the supporting cytoskeleton is provided by the brush border found on the apex 1); recordings ended once the bead escaped from the trap. Under of intestinal epithelial cells (7). The brush border functions as the these conditions we observed that the slope of the membrane primary site for nutrient absorption and consists of up to 1,000 force-extension curve is significantly higher in WT brush borders Ϯ Ϯ microvilli, protrusions that increase apical membrane surface relative to Myo1a KO samples (0.43 0.01 vs. 0.05 0.01 area and release vesicles into the intestinal lumen (7, 8). Each pN/nm, respectively; Fig. 1E). This approach also revealed that is supported by a parallel bundle of actin filaments Myo1a limits the maximum length of membrane tether that we that enables this structure to extend several microns from the cell were able to extract during these experiments (WT, 3.5 Ϯ 0.3 vs. surface (7). To stabilize this convoluted morphology, epithelial KO, 7.8 Ϯ 2.0 ␮m; Fig. 1F). Because these measurements were cells must furnish the brush border with high levels of mem- performed in the absence of active membrane trafficking or brane-cytoskeleton adhesion energy. One candidate molecule other potentially confounding subcellular activities, they clearly for carrying out this task is myosin-1a (Myo1a), a monomeric indicate that Myo1a makes a direct contribution to the mechan- actin-based motor that is present at high levels in the ical stability of the brush border apical membrane. microvillus and is known to bind directly to phospholipids by virtue of its basic C-terminal tail homology 1 (TH1) domain (9, 10). Myo1a KO mice exhibit large apical membrane herniations Author contributions: R.N. and M.J.T. designed research; R.N. performed research; R.N., R.E.M., and M.J.T. contributed new reagents/analytic tools; R.N. and M.J.T. analyzed data; that are morphologically similar to ‘‘blebs’’ (11, 12). Because and R.N. and M.J.T. wrote the paper. blebs represent complete delamination of membrane from the The authors declare no conflict of interest. actin cytoskeleton, these results suggest that Myo1a makes a This article is a PNAS Direct Submission. major contribution to membrane-cytoskeleton adhesion in the 1To whom correspondence should be addressed at: Department of Cell and Developmental brush border. While previous studies have suggested a role for Biology, Vanderbilt University Medical Center, 3130 Medical Research Building III, 465 21st class I myosins in the regulation of cortical stiffness (i.e., whole Avenue South, Nashville, TN 37232-8240. Email: [email protected]. cell deformability) of Dictyostelium discoideum cells (13), the This article contains supporting information online at www.pnas.org/cgi/content/full/ role of class I myosins in the control of membrane tension has 0901641106/DCSupplemental.

11972–11977 ͉ PNAS ͉ July 21, 2009 ͉ vol. 106 ͉ no. 29 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901641106 Downloaded by guest on October 1, 2021 Fig. 1. (A) Confocal micrograph of a membrane tether pulled from a single isolated brush border labeled with Alexa488-Concanavalin A. Image is inverted and contrast enhanced to enable visualization of the extremely dim membrane tether. (B) Phalloidin signal from the isolated brush border shown in a reveals that the tether is devoid of F-actin. The position of the trapped bead and membrane tether are indicated; bars in A and B are 2 ␮m. (C) Merge of images from A and B (shown without contrast enhancement) demonstrates the colocalization of membrane (green) and F-actin (red) signals; panel on the right shows the orientation of microvillar actin bundles in this structure. (D) Cartoon depicting the polarity of microvillar actin bundles of the brush border imaged in A–C.(E) Force-extension records for membrane tethers extracted from WT (green) and Myo1a KO (red) brush borders. Linear fitting of raw data over the first ␮mof extension yields spring constants of 0.43 Ϯ 0.01 pN/nm (n ϭ 6, R2 ϭ 0.99) and 0.05 Ϯ 0.01 pN/nm (n ϭ 7, R2 ϭ 0.92) for WT and KO brush borders, respectively. (F) Box-plots of maximum tether lengths demonstrate that Myo1a KO brush borders released significantly longer tethers (7.8 Ϯ 2.0 ␮m; red) relative to WT controls (3.50 Ϯ 0.34 ␮m; green). *P Ͻ 0.05.

Myo1a Controls TApp in Living Epithelial Cells. We next sought to using 2 methods: (1) expression of an EGFP-tagged Myo1a-TH1 determine whether Myo1a plays a role in regulating apical dominant negative construct, which disrupts the targeting of membrane mechanics in the context of living, polarized epithe- endogenous Myo1a and gives rise to cellular phenotypes similar lial cells. In intact cells, the force exerted by a membrane tether to those observed in Myo1a KO mice (11, 18), and (2) expression held at constant length is directly related to the level of apparent of an EGFP-tagged full length Myo1a construct to supplement membrane tension (6). Importantly, tether formation in this case the population of endogenous Myo1a (19) (Fig. S3). Strikingly, is a reversible process; after forming a tether, release of the expression of the EGFP-Myo1a-TH1 dominant negative in trapped microsphere allows the cell to resorb the extracted NGI3 cells dramatically reduced the force observed during the membrane. We carried out tether force measurements using the tethered phase of individual records, relative to cells expressing colonic adenocarcinoma cell line, NGI3 (16). Upon differenti- EGFP as a negative control (17.9 Ϯ 5.1 vs. 32.1 Ϯ 4.6 pN; Figs. ation, NGI3 cells express endogenous Myo1a and build an 2A and B). In contrast, expressing EGFP-Myo1a in NGI3 cells elaborate brush border with densely packed microvilli. For these gave rise to an increase in tether force relative to control cells experiments, an optical trap was used to capture a ConA-coated (42.5 Ϯ 4.2 pN; Fig. 2A and B). Thus, in the context of live bead and bring it into contact with a surface-adsorbed NGI3 cell epithelial cells, Myo1a enables the apical membrane to resist to enable binding as described above. To form a membrane deformation (i.e., tether formation) and makes a substantial tether, the cell was translated away from the trapped bead at a contribution to apparent membrane tension (TApp). constant rate of 1 ␮m/s. Tethers were pulled to a length of 5 ␮m as our initial experiments with NGI3 cells revealed that tether Myo1a Contributes to Membrane-Cytoskeleton Adhesion (␥) in Living force is independent of length in this regime of extension (Fig. Epithelial Cells and Fibroblasts. The results observed in NGI3 cells S2), in a manner similar to previous results with other cell lines could be explained in 1 of 2 ways: (1) Myo1a could have a direct (17). To probe the contribution of Myo1a to apparent membrane impact on membrane surface tension (TM), or (2) Myo1a could tension, we perturbed the endogenous population of this motor make a significant contribution to membrane-cytoskeleton ad- CELL BIOLOGY

Fig. 2. (A) Representative tether force records from NGI3 cells transfected with EGFP (green), EGFP-Myo1a-TH1 (red), or EGFP-Myo1a (blue). The region of the record marked as ‘‘Tethered’’ corresponds to the quasi-stable equilibrium force achieved after piezo-stage translation has stopped and tether formation is complete. (B) Bar graphs of the average force observed during the tethered phase of individual records reveal that EGFP-Myo1a-TH1 dominant-negative expression lowers tether force (17.9 Ϯ 5.1 pN, n ϭ 14), while expression of EGFP-Myo1a increases tether force (42.5 Ϯ 4.2 pN, n ϭ 11) relative to EGFP-expressing control cells (32.1 Ϯ 4.6 pN, n ϭ 15). *P Ͻ 0.05, **P Ͻ 0.005. (C) Model representation of the correspondence between tether force measurements and the molecular level perturbations induced by expressing EGFP-Myo1a or EGFP-Myo1a-TH1, relative to the EGFP negative control.

Nambiar et al. PNAS ͉ July 21, 2009 ͉ vol. 106 ͉ no. 29 ͉ 11973 Downloaded by guest on October 1, 2021 Fig. 3. (A) Representative multiple tether force records from NGI3 cells transfected with EGFP (green) or EGFP-Myo1a-TH1 (red). Arrows indicate the beginning of the tethered phase in each case. Rapid drops in force during the tethered phase correspond to tether coalescence events. (B) Multiple representative examples of the tethered phases from records of NGI3 cells expressing EGFP (green) and EGFP-Myo1a-TH1 (red). Data were plotted with a fixed offset of 25 pN between all records. Visual inspection reveals that EGFP-Myo1a-TH1 records contain fewer observable coalescence events relative to EGFP control records of the same duration. (C) Box plots show the mean number of coalescence events or ‘‘steps’’ observed in EGFP or EGFP-Myo1a-TH1 records; cells expressing the TH1 dominant negative demonstrate significantly fewer coalescence events, relative to control cells (*P Ͻ 0.05). (D) Box plots of the force-time integrals calculated from the tethered phase of individual records; EGFP-Myo1a-TH1 expressing cells demonstrate significantly lower force-time integrals relative to EGFP-expressing controls (**P Ͻ 0.001). (E) Ensemble averages of tethered phase data from NGI3 cells transfected with EGFP (green) and EGFP-Myo1a-TH1 (red); open circles show average values at 0.3-s intervals; solid lines are single exponential fits to averaged data. Similar force decay kinetics were observed for EGFP (0.12 Ϯ 0.01 sϪ1, n ϭ 28, R2 ϭ 0.99) and EGFP-Myo1a-TH1 (0.13 Ϯ 0.01 sϪ1, n ϭ 30, R2 ϭ 0.98), respectively. (F) Model of the mechanism underlying the formation of multiple tethers in cells expressing EGFP (negative control) or EGFP-Myo1a-TH1 (dominant negative). These cartoons represent ‘‘snap shots’’ in the records shown in A, taken at the beginning of the tethered phases at the time point indicated by the black arrows.

hesion energy (␥) by physically linking the membrane to the actin membrane tether coalescence (22, 24). Similar to previous cytoskeleton. With its combined membrane- and actin-binding studies of multiple tether mechanics (20, 25), observed force activities, Myo1a is ideally suited for mediating to membrane- steps appear as integer multiples of the force measured for single cytoskeleton adhesion; decreasing or increasing the number of tethers (Fig. S5B and C). Intriguingly, visual inspection of force functional Myo1a molecules per unit area of membrane with the records revealed that expression of the EGFP-Myo1a-TH1 expression of EGFP-Myo1a-TH1 or EGFP-Myo1a, respectively, dominant negative significantly impaired the cell’s ability to would give rise to a corresponding reduction or elevation in stabilize multiple tethers. This was indicated by 2 important adhesion energy, and thus apparent membrane tension (Fig. 2C). observations. First, the tethered phase of records from TH1- To further test this hypothesis, we performed tether force expressing cells appeared to start at a lower level of force (Fig. measurements under conditions that favored the formation of 3A). We confirmed this by calculating force-time integrals across multiple membrane tethers (20, 21). When multiple membrane the tethered phase of individual records. EGFP-Myo1a-TH1 tethers are simultaneously pulled from the same local region of expression significantly reduced the mean force-time integral, membrane, they demonstrate a tendency to coalesce into a single suggesting a reduction in the total number of tethers that tether in the absence of ‘‘pinning’’ forces (22), for example, contribute to force during the tethered phase (Fig. 3D). Second, forces provided by molecular links to the underlying cytoskele- when the number of observable steps (i.e., coalescence events) ton. Thus, assaying the number of tethers formed from a single was tallied from force records, TH1-expressing cells demon- microsphere/cell encounter provides a read-out on the density of strated a significantly reduced average number of events per molecular contacts between the membrane and cytoskeleton. record (Fig. 3B and C). Increasing the contact area between the microsphere and cell, or As discussed above, the force steps observed during multiple increasing the loading rate (by raising trap stiffness) biased tether events are integer multiples of a unitary tether force (ϳ30 events toward the formation of multiple tethers (23) (Fig. S4A pN in the case of NGI3 cells, Fig. 2 and Fig. S5B), suggesting that and B). Multiple tether formation was confirmed using DIC each step represents the same underlying physical process, that microscopy (Fig. S5A). The presence of multiple tethers was also is, tether coalescence. Because the coalescence of membrane indicated by the appearance of ‘‘staircases’’ during the tethered tethers is due to the failure of bonds that link the membrane and phase of force records (Fig. 3A). The rapid drops or ‘‘steps’’ underlying cytoskeleton, we expect the irreversible transition between discrete plateaus in force are the result of adjacent from n tethers to n-1 tethers to proceed as a first-order process

11974 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901641106 Nambiar et al. Downloaded by guest on October 1, 2021 Fig. 4. (A) Representative tether force records for NIH 3T3 fibroblasts expressing EGFP (green) or 1 of 5 different EGFP-tagged class I myosins (as labeled on the plot). (B) Box plots of force-time integrals calculated over the tethered phases of individual records from NIH 3T3 cells expressing the various class I myosin constructs (*P Ͻ 0.05, **P Ͻ 0.001). Cells expressing myosin-I constructs uniformly demonstrate higher force-time integrals relative to EGFP expressing controls. (C) Ensemble averages of tethered phases from records of NIH 3T3 cells transfected with various class I myosin constructs (open circles); solid lines show single Ϫ1 2 Ϫ1 exponential fits to the data. Rate constants obtained from exponential fits are as follows: EGFP, 0.04 Ϯ 0.01 s , n ϭ 23, R ϭ 0.97; EGFP-Myo1a, 0.17 Ϯ 0.01 s , 2 Ϫ1 2 Ϫ1 2 Ϫ1 2 n ϭ 21, R ϭ 0.99; EGFP-Myo1b, 0.06 Ϯ 0.01 s , n ϭ 15, R ϭ 0.97; EGFP-Myo1c, 0.13 Ϯ 0.01 s , n ϭ 10, R ϭ 0.91; EGFP-Myo1d, 0.20 Ϯ 0.01 s , n ϭ 9, R ϭ 0.96; Ϫ1 2 EGFP-Myo1e, 0.28 Ϯ 0.01 s , n ϭ 5, R ϭ 0.98.

with a rate equal to approximately 1/lifetime of a single tether vs. 0.12 Ϯ 0.01 sϪ1, respectively), which normally express signif- (26). Indeed, ensemble averaging of the tethered phases from icant levels of endogenous Myo1a. This kinetic similarity sug- individual EGFP and EGFP-Myo1a-TH1 records revealed sin- gests that our tether force measurements are in fact probing the gle-exponential decays in force for both data sets (Fig. 3E). Fits mechanical contributions made by Myo1a in the case of both to these data revealed comparable kinetics (EGFP, 0.12 Ϯ 0.01 experiments. sϪ1; EGFP-Myo1a-TH1, 0.13 Ϯ 0.01 sϪ1) indicating that a similar molecular process controls the rate of tether coalescence in Control of Membrane Mechanics May Be a Class-Wide Function for EGFP and EGFP-Myo1a-TH1-expressing cells. We propose Class I Myosins. The results summarized to this point indicate that that Myo1a plays a role in stabilizing multiple membrane tethers Myo1a controls apparent membrane tension (TApp) by contrib- in both cases. However, in cells expressing the dominant nega- uting to membrane-cytoskeleton adhesion (␥). Given that all 8 tive, a large fraction of the endogenous Myo1a population is vertebrate class I myosins contain a basic TH1 domain (27), displaced from the plasma membrane; this reduces its effective which mediates interactions with cellular membranes (28), we contribution to membrane-cytoskeleton adhesion and ultimately sought to determine whether other myosin-I isoforms could also impairs the cell’s ability to form and stabilize multiple tethers contribute to membrane-cytoskeleton adhesion. To this end, we (Fig. 3F). expressed EGFP-tagged versions of 3 other short-tailed (Myo1b, Based on the model outlined above, one prediction is that the Myo1c, and Myo1d) and 1 long-tailed (Myo1e) class I myosins expression of EGFP-Myo1a in NGI3 cells should enhance the in NIH 3T3 cells (Fig. S6) and probed their impact on multiple cell’s capacity for stabilizing multiple tethers. While we at- tether formation as described above. Inspection of raw data tempted multiple tether experiments with NGI3 cells expressing records qualitatively revealed that regardless of the isoform, EGFP-Myo1a, our ability to extract tethers from these cells was class I myosin expression increased the force measured during dramatically decreased. If Myo1a does contribute to membrane- the tethered phase relative to EGFP-expressing controls (Fig. cytoskeleton adhesion (␥), then this observation might reflect 4A). This was confirmed through quantitative analysis of mul- the expected outcome. Because NGI3 cells express endogenous tiple tether records by calculating average force-time integrals Myo1a, transfection with EGFP-Myo1a creates an over- (Fig. 4B) and producing ensemble averages of data (Fig. 4C) for expression scenario that could give rise to exaggerated mem- each construct as outlined above (see Fig. 3). Of note here is the brane-cytoskeleton adhesion. We suspect that the extraction of fact that Myo1e, the only long-tailed isoform included in our multiple tethers is precluded in this case, as the forces required analysis, had the most dramatic impact on multiple tether are beyond the upper limits of our optical trap. In light of this formation. The uniform increase in multiple tether formation possibility, we examined the impact of EGFP-Myo1a expression observed with the expression of different myosin I isoforms, on multiple tether formation from cells that express low levels of suggests that membrane-cytoskeleton adhesion and the control endogenous Myo1a. For these experiments, we used NIH 3T3 of plasma membrane tension may be general functions for all fibroblasts, which do not express detectable Myo1a and exhibit vertebrate class I myosins. a low apparent membrane tension (ϳ25% of TApp for NGI3, Fig. S4C) (5). Multiple tether force measurements in NIH 3T3 cells Discussion produced step-containing records as observed in NGI3 cells Experiments performed in multiple eukaryotic model systems

described above. Inspection of raw data and analysis of force- have implicated class I myosins in various aspects of membrane- CELL BIOLOGY time integrals revealed that expression of EGFP-Myo1a in NIH related events including phagocytosis (29–33), endocytosis (34– 3T3 cells produces a modest increase in the force observed 37), exocytosis (38, 39), and membrane recycling (40). Although during the tethered phase (Fig. 4, royal blue data). These results our current data set does not allow us to rule out the possibility show that Myo1a expression is capable of increasing membrane- that perturbations in membrane trafficking may contribute to cytoskeleton adhesion outside the context of the polarized the changes in membrane tension observed in our experiments, cytoskeleton found in NGI3 cells. our results do provide strong support for a model where class I Interestingly, the force decay kinetics in NIH 3T3 cells ex- myosins play a direct role in the control of membrane tension, pressing EGFP-Myo1a were comparable to those observed in by contributing to adhesion between the plasma membrane and experiments with NGI3 cells (Fig. 4C vs. Fig. 3E; 0.17 Ϯ 0.01 sϪ1 underlying actin cytoskeleton. Indeed, mechanical measure-

Nambiar et al. PNAS ͉ July 21, 2009 ͉ vol. 106 ͉ no. 29 ͉ 11975 Downloaded by guest on October 1, 2021 ments with isolated brush borders revealed that Myo1a increases pear to be tuned to enable these motors to contribute to apical membrane force-extension stiffness approximately 10-fold membrane tension (44). Recent single molecule studies indicate (Fig. 1E). Because these experiments were performed in the that the activity of myosin-1b is exquisitely sensitive to opposing absence of ATP, potentially confounding processes such as external load (45). These studies show that the rate of ADP membrane trafficking were not active during these in vitro release and thus detachment from actin slows down approxi- measurements and had little impact on the observed mechanical mately 100-fold in response to loads as small as 2 pN (45). responses. This model finds additional support when we consider Load-dependent kinetics may enable class I myosins to function that disrupting Myo1a function in polarized epithelial (NGI3) in membrane/cytoskeleton adhesion by allowing them to remain cells reduced the mean tether force by approximately 50% (Fig. strongly bound to F-actin for long periods, without hydrolyzing 2A and B). Apparent membrane tension (TApp), considered to ATP. be the sum of in-plane tension (Tm) and membrane/cytoskeleton Although the detailed kinetics of myosin-I/actin interactions adhesion (␥), is directly related to the tether force (FTether): TApp are in many cases well-characterized (44), less is known about 2 2 ϭ Tm ϩ ␥ ϭ (FTether) /(8B␲ ), where B is the membrane bending the mechanism underlying myosin-I membrane binding. While stiffness (41). Thus, perturbation of Myo1a reduced apparent it was established many years ago that Myo1a binds to acidic membrane tension by approximately 70%. This value ap- phospholipids via its basic TH1 domain (10), more recent proaches previously published estimates that attribute over 75% studies have revealed that vertebrate and Acanthamoeba class of apparent membrane tension to membrane-cytoskeleton ad- I myosin TH1 domains contain a PH motif able to bind tightly hesion (6). Finally, analysis of multiple tether formation provides to highly charged, acidic phosphoinositides such as PIP2 (46, some of the most direct support for this model. Expression of the 47). These studies may help explain earlier biophysical data Myo1a TH1 dominant negative decreased the ability of NGI3 implicating PIP2 in membrane/cytoskeleton adhesion (48). cells to support and stabilize multiple membrane tethers, However, PIP2 is estimated to comprise Ͻ1% of total phos- whereas over-expression of Myo1a or other class I myosins pholipid found in the inner leaflet of the plasma membrane stabilized multiple membrane tethers (Figs. 3 and 4). Because (49). Thus, in domains such as the brush border, where the ability to form multiple tethers is directly linked to the membrane/cytoskeleton adhesion must be high to maintain a density of molecular contacts between the membrane and complex morphology, higher abundance lipids (e.g., phospha- cytoskeleton, these results tell us that class I myosins are tidylserine) and alternate, unexplored lipid binding motifs important players in mediating these interactions. Thus, the within the TH1 domain are likely to play a role. Finally, results presented here strongly support a model where class I because TH1 domains have been identified in myosin-I myosins play a direct role in the control of membrane tension, from the earliest eukaryotes (27), the control of membrane by contributing to adhesion between the plasma membrane and tension may represent an ancient and conserved function for underlying actin cytoskeleton. these molecular motors. The mechanical measurements presented here provide a physical explanation for the phenotypes observed in the Myo1a Materials and Methods KO mouse (11). Among the most striking defects observed in Optical Trap Instrumentation. Our optical trap is built around a single-mode this model are herniations of apical membrane that extend from diode-pumped solid-state Nd:YVO4 laser (LG Laser Technologies; TEM00,3W, the apical surface of KO enterocytes. In most cell types cytosolic ␭ ϭ 1064 nm) that is coupled to a Nikon TE-2000-U inverted light microscope fluid pressure, created by myosin-II powered contractility in the via optics that are housed in central unit from Molecular Machines & Indus- cell cortex, exerts a positive (i.e., outward) force on the plasma tries. In addition to the laser head, the central unit contains beam- membrane (42). In the enterocyte, the high levels of membrane- conditioning optics, z axis focusing lenses, and 2 galvanometer-mounted cytoskeleton adhesion provided by the microvillar population of mirrors for the control of beam position. We used a Nikon PlanFluor 100x/1.3 Myo1a function to counter cytosolic pressure so that the brush lens (72% transmission in the IR) to focus the laser and form a trap at the focal place. A motorized X-Y scanning stage (Marzhauser) was used for course border can stabilize the enormous quantity of plasma membrane control of sample position. A piezoelectric stage insert (Mad City Labs) pro- packed into this domain. vided high-resolution position control with subnanometer accuracy. Scanning In addition to providing access to information about mem- stage position, laser power, and laser focal depth (z axis trap position) were all brane-cytoskeleton adhesion, the multiple tether experiments under computer via software provided by MMI. Images of trapped particles described here may provide important mechanistic informa- were captured with a Sony Exwave HAD color CCD using a National Instru- tion on the formation of ‘‘tethers’’ under normal physiological ments PCI-1410 image acquisition card with custom software written in Lab- conditions. As an example, leukocytes rolling along endothe- VIEW 8.5. Time-lapse images acquired at video rate were used to obtain the lium extrude multiple membrane tethers to stabilize their position of trapped particles with software developed by Carter et al. (15). rolling velocities, ultimately enabling arrest and extravasation Trap stiffness calibration was performed by measuring the excursion of a (25). Thus, one goal for future studies will be to determine trapped bead in response to different viscous drag forces applied by moving the flow chamber with the piezoelectric stage. Stokes’ law (6␲␩rv ϭ kTrap x; whether the class I myosins expressed in leukocytes, play a role where v is solution velocity, ␩ is coefficient of solution viscosity, r is the radius in the formation and stabilization of these important mem- of the microsphere, and x is microsphere displacement from trap center) was brane structures. used to calculate the value of kTrapfor our experiments (Fig. S1). While the importance of the actin cytoskeleton in shaping the plasma membrane and its mechanical properties is well estab- Brush Border Isolation and Manipulation. Brush borders were isolated using lished (14), the results described here show that actin-based previously described protocol (50). All procedures involving animals were motors, and specifically class I myosins, play a role in controlling performed under the protocols prescribed by the Vanderbilt University Med- the mechanical interactions between these 2 systems. Class I ical Center Institutional Animal Care and Use Committee. For membrane molecules are ideal candidates for fulfilling this function within tether studies, brush borders were typically transferred into a flow cell assem- cells. Cryo-electron microscopy studies have established that bled with a glass slide, a coverslip and 2 pieces of doubled-sided tape. Several Myo1a is an extended molecule arranged with the actin-binding volumes of buffer were applied to the flow cell to flush out brush borders that were only loosely anchored to the glass; tether experiments were then carried motor domain at 1 end, and the putative lipid interacting domain out in 75 mM KCl, 10 mM imidazole, 1 mM EGTA, 2.5 mM MgCl2, and 0.01% at the other (43). This domain arrangement is well suited for the Na-Azide, pH 7.2. bivalent crosslinking of plasma membrane to actin filaments, while maintaining an approximate 15-nm (projected length of Cell Culture and Transfections. NGI3 and NIH 3T3 cells were cultured on Myo1a) gap between these 2 compartments. In addition to coverslips in 6-well plates at 37°C, with 5% CO2. Culture medium consisted domain organization, specific mechanochemical properties ap- of DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hy-

11976 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901641106 Nambiar et al. Downloaded by guest on October 1, 2021 Clone) and 2 mM glutamine (GibcoBRL). Transfections were carried out in ACKNOWLEDGMENTS. We thank members of the Tyska Laboratory for 6-well plates with 6–8 ␮g DNA per 2.5 mL plating media using the reagent helpful suggestions and Dr. Stefan Niehren of Molecular Machines & Lipofectamine2000 (Invitrogen). Cells were assayed 2–3 days post transfec- Industries for outstanding support. This work was supported in part by tion. In a typical experiment, cell-coated coverslips were assembled into a the Vanderbilt Digestive Diseases Research Center P30 DK-058404, the Vanderbilt University Medical Center Training Program in Developmental flow chamber using double-sided tape and a glass slide. For all experi- Biology (R.E.M.), a predoctoral fellowship from the American Heart Asso- ments, cells were incubated in CO2-independent medium (Invitrogen) at ciation (R.E.M.), a postdoctoral fellowship from the American Heart Asso- 37°C with an objective heater controlled by a TC-124 temperature control- ciation (R.N.), and a National Institutes of Health Grant R01-DK075555 (to ler (Warner Instruments). M.J.T.).

1. Dai J, Ting-Beall HP, Sheetz MP (1997) The secretion-coupled endocytosis correlates 27. Richards TA, Cavalier-Smith T (2005) Myosin domain evolution and the primary diver- with membrane tension changes in RBL 2H3 cells. J Gen Phys 110:1–10. gence of eukaryotes. Nature 436:1113–1118. 2. Apodaca G (2002) Modulation of membrane traffic by mechanical stimuli. Am J Physiol 28. Coluccio LM (1997) Myosin I. Am J Physiol 273:C347–359. 282:F179–190. 29. Jung G, Wu X, Hammer JA, 3rd (1996) Dictyostelium mutants lacking multiple classic myosin I 3. Togo T, Krasieva TB, Steinhardt RA (2000) A decrease in membrane tension precedes isoforms reveal combinations of shared and distinct functions. J Cell Biol 133:305–323. successful cell-membrane repair. Mol Biol Cell 11:4339–4346. 30. Voigt H, Olivo JC, Sansonetti P, Guillen N (1999) Myosin IB from Entamoeba histolytica 4. Sheetz MP, Dai J (1996) Modulation of membrane dynamics and cell motility by is involved in phagocytosis of human erythrocytes. J Cell Sci 112:1191–1201. membrane tension. Trends in Cell Biol 6:85–89. 31. Schwarz EC, Neuhaus EM, Kistler C, Henkel AW, Soldati T (2000) Dictyostelium myosin 5. Raucher D, Sheetz MP (2000) Cell spreading and lamellipodial extension rate is regu- IK is involved in the maintenance of cortical tension and affects motility and phago- lated by membrane tension. J Cell Biol 148:127–136. cytosis. J Cell Sci 113:621–633. 6. Sheetz MP (2001) Cell control by membrane-cytoskeleton adhesion. Nat Rev Mol Cell 32. Marion S, Wilhelm C, Voigt H, Bacri JC, Guillen N (2004) Overexpression of myosin IB in Biol 2:392–396. living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis. 7. Mooseker MS (1985) Organization, chemistry, and assembly of the cytoskeletal appa- J Cell Sci 117:3271–3279. ratus of the intestinal brush border. Ann Rev Cell Biol 1:209–241. 33. Durrwang U, et al. (2006) Dictyostelium myosin-IE is a fast involved in 8. McConnell RE, Tyska MJ (2007) Myosin-1a powers the sliding of apical membrane along phagocytosis. J Cell Sci 119:550–558. microvillar actin bundles. J Cell Biol 177:671–681. 34. Yamashita RA, May GS (1998) Constitutive activation of endocytosis by mutation of 9. Mooseker MS, Coleman TR (1989) The 110-kD protein- complex of the intesti- myoA, the myosin I of Aspergillus nidulans. J Biol Chem 273:14644–14648. nal microvillus (brush border myosin I) is a mechanoenzyme. J Cell Biol 108:2395–2400. 35. Durrbach A, Raposo G, Tenza D, Louvard D, Coudrier E (2000) Truncated brush border 10. Hayden SM, Wolenski JS, Mooseker MS (1990) Binding of brush border myosin I to myosin I affects membrane traffic in polarized epithelial cells. Traffic 1:411–424. phospholipid vesicles. J Cell Biol 111:443–451. 36. Sokac AM, Schietroma C, Gundersen CB, Bement WM (2006) Myosin-1c couples assem- 11. Tyska MJ, et al. (2005) Myosin-1a is critical for normal brush border structure and bling actin to membranes to drive compensatory endocytosis. Dev Cell 11:629–640. composition. Mol Biol Cell 16:2443–2457. 37. Krendel M, Osterweil EK, Mooseker MS (2007) Myosin 1E interacts with synaptojanin-1 12. Charras GT, Hu CK, Coughlin M, Mitchison TJ (2006) Reassembly of contractile actin and and is involved in endocytosis. FEBS Lett 581:644–650. cortex in cell blebs. J Cell Biol 175:477–490. 38. Bose A, et al. (2004) Unconventional myosin Myo1c promotes membrane fusion in a 13. Dai J, Ting-Beall HP, Hochmuth RM, Sheetz MP, Titus MA (1999) Myosin I contributes regulated exocytic pathway. Mol Cell Biol 24:5447–5458. to the generation of resting cortical tension. Biophys J 77:1168–1176. 39. Schietroma C, et al. (2007) A role for myosin 1e in cortical granule exocytosis in Xenopus 14. Dai J, Sheetz MP (1995) Mechanical properties of neuronal growth cone membranes oocytes. J Biol Chem 282:29504–29513. studied by tether formation with laser optical tweezers. Biophys J 68:988–996. 40. Neuhaus EM, Soldati T (2000) A myosin I is involved in membrane recycling from early 15. Carter BC, Shubeita GT, Gross SP (2005) Tracking single particles: A user-friendly endosomes. J Cell Biol 150:1013–1026. quantitative evaluation. Phys Biol 2:60. 41. Hochmuth FM, Shao JY, Dai J, Sheetz MP (1996) Deformation and flow of membrane 16. Tian JQ, Quaroni A (1999) Dissociation between growth arrest and differentiation in into tethers extracted from neuronal growth cones. Biophys J 70:358–369. Caco-2 subclone expressing high levels of sucrase. Am J Physiol 276:G1094–1104. 42. Charras G, Paluch E (2008) Blebs lead the way: How to migrate without lamellipodia. 17. Raucher D, Sheetz MP (1999) Characteristics of a membrane reservoir buffering mem- brane tension. Biophys J 77:1992–2002. Nat Rev Mol Cell Biol 9:730–736. 18. Tyska MJ, Mooseker MS (2004) A role for myosin-1A in the localization of a brush 43. Jontes JD, Milligan RA (1997) Three-dimensional structure of Brush Border Myosin-I at border disaccharidase. J Cell Biol 165:395–405. approximately 20 A resolution by electron microscopy and image analysis. J Mol Biol 19. Tyska MJ, Mooseker MS (2002) Myo1a (brush border myosin I) dynamics in the brush 266:331–342. border of LLC-PK1-CL4 cells. Biophys J 82:1869–1883. 44. De La Cruz EM, Ostap EM (2004) Relating biochemistry and function in the myosin 20. Sun M, et al. (2005) Multiple membrane tethers probed by atomic force microscopy. superfamily. Curr Opin Cell Biol 16:61–67. Biophys J 89:4320–4329. 45. Laakso JM, Lewis JH, Shuman H, Ostap EM (2008) Myosin I can act as a molecular force 21. Hosu BG, Sun M, Marga F, Grandbois M, Forgacs G (2007) Eukaryotic membrane tethers sensor. Science 321:133–136. revisited using magnetic tweezers. Phys Biol 4:67. 46. Hokanson DE, Laakso JM, Lin T, Sept D, Ostap EM (2006) Myo1c binds phosphoinositi- 22. Dere´nyi I, Ju¨licher F, Prost J (2002) Formation and interaction of membrane tubes. Phys des through a putative pleckstrin homology domain. Mol Biol Cell 17:4856–4865. Rev Lett 88:238101. 47. Brzeska H, Hwang KJ, Korn ED (2008) Acanthamoeba myosin IC colocalizes with 23. Ramachandran V, Williams M, Yago T, Schmidtke DW, McEver RP (2004) Dynamic phosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the high alterations of membrane tethers stabilize leukocyte rolling on P-selectin. Proc Nat concentration of negative charge. J Biol Chem 283:32014–32023. Acad of Sci 101:13519–13524. 48. Raucher D, et al. (2000) Phosphatidylinositol 4,5-bisphosphate functions as a second 24. Cuvelier D, Derenyi I, Bassereau P, Nassoy P (2005) Coalescence of membrane tethers: messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221–228. Experiments, theory, and applications. Biophys J 88:2714–2726. 49. Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev 25. Xu G, Shao JY (2005) Double tether extraction from human neutrophils and its Mol Cell Biol 9:99–111. comparison with CD4ϩ T lymphocytes. Biophys J 88:661–669. 50. Howe CL, Mooseker MS (1983) Characterization of the 110-kdalton actin-calmodulin-, 26. Evans E (2001) Probing the relation between force–lifetime–and chemistry in single and membrane-binding protein from microvilli of intestinal epithelial cells. J Cell Biol molecular bonds. Annu Rev Biophys Biomol Struct 30:105–128. 97:974–985. CELL BIOLOGY

Nambiar et al. PNAS ͉ July 21, 2009 ͉ vol. 106 ͉ no. 29 ͉ 11977 Downloaded by guest on October 1, 2021