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Myosin IIA Interacts with the Spectrin-Actin Membrane Skeleton to Control Red Blood Cell Membrane Curva- Ture and Deformability,” by Alyson S

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Myosin IIA Interacts with the Spectrin-Actin Membrane Skeleton to Control Red Blood Cell Membrane Curva- Ture and Deformability,” by Alyson S Correction CELL BIOLOGY Correction for “Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curva- ture and deformability,” by Alyson S. Smith, Roberta B. Nowak, Sitong Zhou, Michael Giannetto, David S. Gokhin, Julien Papoin, Ionita C. Ghiran, Lionel Blanc, Jiandi Wan, and Velia M. Fowler, which was first published April 2, 2018; 10.1073/pnas.1718285115 (Proc Natl Acad Sci USA 115:E4377–E4385). The authors note that the grant number UL1 TR00114 should instead appear as TL1 TR001113. Published under the PNAS license. Published online June 25, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1809742115 CORRECTION www.pnas.org PNAS | July 3, 2018 | vol. 115 | no. 27 | E6385 Downloaded by guest on September 26, 2021 Myosin IIA interacts with the spectrin-actin membrane PNAS PLUS skeleton to control red blood cell membrane curvature and deformability Alyson S. Smitha,1, Roberta B. Nowaka,1, Sitong Zhoub,c,d, Michael Giannettob,c,d, David S. Gokhina, Julien Papoine, Ionita C. Ghiranf, Lionel Blance,g,h, Jiandi Wanb,c,d, and Velia M. Fowlera,2 aDepartment of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037; bMicrosystems Engineering, Rochester Institute of Technology, Rochester, NY 14623; cDepartment of Biomedical Engineering, University of Rochester, Rochester, NY 14623; dCenter for Translational Neuromedicine, University of Rochester Medical Center, Rochester, NY 14623; eCenter for Autoimmune, Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute for Medical Research, Manhasset, NY 11030; fDepartment of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115; gDepartment of Molecular Medicine, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11030; and hDepartment of Pediatrics, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11030 Edited by Vann Bennett, Duke University Medical Center, Durham, NC, and approved March 14, 2018 (received for review October 19, 2017) The biconcave disk shape and deformability of mammalian RBCs membrane domains of ion channels, pumps, or cell adhesion mol- rely on the membrane skeleton, a viscoelastic network of short, ecules that confer complex signaling, cell interactions, and mechanical membrane-associated actin filaments (F-actin) cross-linked by resilience to the plasma membrane (8–10). long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) Although researchers first documented the biconcave disk motors exert force on diverse F-actin networks to control cell shape of RBCs nearly 200 years ago (11), numerous unanswered shapes, but a function for NMII contractility in the 2D spectrin–F- questions remain regarding the forces that generate and main- actin network of RBCs has not been tested. Here, we show that tain this unique cellular morphology (5, 12, 13). Nonmuscle RBCs contain membrane skeleton-associated NMIIA puncta, iden- myosin II (NMII), a force-generating motor protein identified tified as bipolar filaments by superresolution fluorescence micros- and purified from RBCs >30 years ago, forms heterohexamers copy. MgATP disrupts NMIIA association with the membrane (termed NMII molecules) of two heavy chains (HC), two regu- CELL BIOLOGY skeleton, consistent with NMIIA motor domains binding to mem- latory light chains (RLC), and two essential light chains (14, 15). brane skeleton F-actin and contributing to membrane mechanical In vitro experiments show that, similar to NMII from other cells, properties. In addition, the phosphorylation of the RBC NMIIA RBC NMII molecules have F-actin–activated MgATPase activity heavy and light chains in vivo indicates active regulation of NMIIA regulated by myosin light chain kinase (MLCK) phosphorylation motor activity and filament assembly, while reduced heavy chain of the RLC and can assemble into bipolar filaments with motor phosphorylation of membrane skeleton-associated NMIIA indi- domains at filament ends (14–16). Each mature human RBC cates assembly of stable filaments at the membrane. Treatment contains ∼6,200 NMII molecules and ∼500,000 actin molecules, of RBCs with blebbistatin, an inhibitor of NMII motor activity, de- resulting in about 80 actin molecules per NMII molecule, similar creases the number of NMIIA filaments associated with the mem- to the ratio in other cells, such as platelets (15–17). NMII in brane and enhances local, nanoscale membrane oscillations, suggesting mature RBCs has been hypothesized to control RBC shapes (15, decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deform- Significance ability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures The biconcave disk shape and deformability of the mammalian similar to the RBC membrane skeleton exist in many metazoan cell RBC are vital to its circulatory function and rely upon a 2D types, these data demonstrate a general function for NMII in controlling viscoelastic spectrin–F-actin network attached to the mem- specialized membrane morphology and mechanical properties through brane. A role for nonmuscle myosin II (NMII) contractility in contractile interactions with short F-actin in spectrin–F-actin networks. generating tension in this network and controlling RBC shape has not been tested. We show that NMIIA forms bi- actomyosin contractility | cytoskeleton | erythrocyte deformability | polar filaments in RBCs, which associate with F-actin at the cell shape | TIRF microscopy membrane. NMIIA motor activity regulates interactions with the spectrin–F-actin network to control RBC biconcave shape and BC biconcave disk shape and deformability provide a maxi- deformability. These results provide a previously undescribed Rmal surface-area-to-volume ratio for optimal gas and ion mechanism for actomyosin force generation at the plasma exchange and enable repeated transit through blood vessels less membrane, and may apply to spectrin–F-actin–based mem- than half their diameter during the ∼120-day RBC lifespan (1–3). brane skeleton networks in other cell types, such as neurons Appropriate levels of RBC deformation also regulate blood flow and polarized epithelial cells. and oxygen delivery via mechanotransductive pathways that re- lease ATP to induce vasodilation and hyperemia (4). These Author contributions: A.S.S., R.B.N., I.C.G., L.B., J.W., and V.M.F. designed research; A.S.S., R.B.N., S.Z., M.G., D.S.G., J.P., I.C.G., and V.M.F. performed research; A.S.S., R.B.N., S.Z., properties all rely upon the membrane skeleton, a 2D quasihex- D.S.G., I.C.G., L.B., J.W., and V.M.F. analyzed data; and A.S.S., L.B., J.W., and V.M.F. wrote agonal network of short (∼37 nm) actin filament (F-actin) nodes the paper. interconnected by ∼200-nm-long, flexible (α1β1)2-spectrin tetra- The authors declare no conflict of interest. mers (5, 6). Molecular genetics, biochemistry, biophysics, and This article is a PNAS Direct Submission. physiology of human and mouse congenital hemolytic anemias have Published under the PNAS license. shown that multiple proteins, which mediate the connectivity of the 1A.S.S. and R.B.N. contributed equally to this work. ’ micrometer-scale 2D network and the network s multipoint at- 2To whom correspondence should be addressed. Email: [email protected]. tachments to the membrane, are critical for RBC shape and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deformability (1, 2, 5, 7). Similar spectrin–F-actin networks are a 1073/pnas.1718285115/-/DCSupplemental. conserved feature of metazoan cells, where they create specialized www.pnas.org/cgi/doi/10.1073/pnas.1718285115 PNAS Latest Articles | 1of9 18, 19), repair local disruptions in the spectrin–F-actin network brane skeleton is a thin, regular, 2D spectrin–F-actin network (20), or remain as a nonfunctional vestige from an earlier and that underlies the membrane and consists of much shorter more motile stage of erythroblast terminal differentiation and (∼37 nm) actin filaments that bind a unique complement of maturation (17), but none of these hypotheses have been experi- proteins (25). Because of these differences in geometry and mentally tested. composition, it is an open question whether NMII can pull on In most nucleated eukaryotic cells, NMII bipolar filaments the short F-actin of the RBC membrane skeleton, or the mem- generate tension to control membrane deformations and cell brane skeleton networks of other metazoan cells, to generate shape by pulling on F-actin in the cortex, a thick, irregular, 3D tension and influence membrane properties, such as curvature network of relatively long (100–600 nm) actin filaments adjacent and mechanics. RBCs are an ideal model system to test this to the plasma membrane (21–24). By contrast, the RBC mem- question, as RBC F-actin is present exclusively in the membrane A 3D reconstruction DEMinimum distance between puncta NMIIA Phalloidin Merge cell 3 200 35 30 cell 1 cell 2 150 25 20 100 15 2μm 10 Puncta/cell 50 % NMIIA puncta % NMIIA 5 B Single optical section 0 0 NMIIA cell 1 cell 2 cell 3 Whole Cell 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 F Minimum distance (μm) GFP NMIIA Tail NMIIA Phalloidin 0.5µm GHGFP GFP GFP NMIIA Tail NMIIA Tail 2μm Merge 200nm C 3D reconstruction NMIIA 1.5 I TIRF J NMIIA Phalloidin Merge 2 1.0 0.5 2μm Puncta/μm 0 0.7μm 0-450nm yellow At membrane Fig. 1. NMIIA localizes as puncta, which likely represent bipolar filaments, throughout each RBC. (A) Three-dimensional reconstruction of a superresolution AiryScan confocal Z-stack of human RBCs immunostained with an antibody to the motor domain of NMIIA (green) and rhodamine-phalloidin for F-actin (red). (B) Higher magnification views of NMIIA motor domain puncta in single XY optical sections from the superresolution AiryScan Z-stack shown in A, showing some puncta at the membrane (arrowheads) and some closely spaced doublets of puncta (arrows).
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