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Control of Cell Membrane Tension by Myosin-I

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Control of Cell Membrane Tension by Myosin-I Control of cell membrane tension by myosin-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 myosins 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 actin cytoskeleton 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 proteins, 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 microvillus 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 protein 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.
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