Compartmentalization of the erythrocyte membrane by the membrane skeleton: intercompartmental hop diffusion of band 3.

Abstract

Molecular Biology of the CellVol. 10, No. 8 ArticlesFree AccessCompartmentalization of the Erythrocyte Membrane by the Membrane Skeleton: Intercompartmental Hop Diffusion of Band 3Michio Tomishige, and Akihiro KusumiMichio Tomishige Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; and Search for more papers by this author, and Akihiro Kusumi Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; and Kusumi Membrane Organizer Project, Exploratory Research on Advanced Technology Organization, Japan Science and Technology Corporation, Nagoya 460-0012, Japan‡Corresponding author. E-mail address: E-mail Address: [email protected].Search for more papers by this authorW. James Nelson, Monitoring EditorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.10.8.2475AboutSectionsView articleSupplemental MaterialView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail View articleINTRODUCTIONRecent developments in microscopic instrumentation and probes have allowed the observation and manipulation of the movement of membrane proteins and lipids in the plasma membrane at the level of single molecules. These experiments are performed by tracking small colloidal gold particles attached to specific membrane proteins and lipids (single particle tracking) (Jacobson et al., 1995; Sheetset al., 1995; Kusumi and Sako, 1996; Saxton and Jacobson, 1997) or by dragging particle–protein complexes along the surface of the plasma membrane using laser tweezers (also termed an “optical trap” or OT) (Edidin et al., 1991; Kusumi and Sako, 1996;Kusumi et al., 1998). In optimal cases, a single protein molecule is attached to a 40-nm-diameter colloidal gold probe (Tomishige et al., 1998). These single molecule experiments have demonstrated that most membrane-spanning proteins do not undergo simple diffusion, but that a significant number are locally slowed or stopped, some are temporarily confined, and others are transported unidirectionally (Kusumi et al., 1993; Sheets et al., 1997; Sako et al., 1998; Simson et al., 1998). Previously, these processes had not been observed, because the methods used, e.g., fluorescence recovery after photobleaching, recorded the ensemble-averaged behavior of a large number of molecules.The underlying cellular mechanisms that induce nonrandom diffusion have remained largely unknown; however, we and others found that properties of the membrane skeleton network fundamentally affect the movement and distribution of certain membrane proteins, such as transferrin receptor, α2-macroglobulin receptor, MHC class I molecules, E-cadherin, and band 3, through corralling and binding effects imposed by the membrane skeleton network (Edidin et al., 1991; Luna and Hitt, 1992; Kusumi et al., 1993;Sako and Kusumi, 1994, 1995; Sako et al., 1998; Tomishigeet al., 1998). The results are particularly clear with band 3 in human erythrocyte ghost membranes, where the specimen consists of a lipid bilayer and its underlying membrane skeleton, which has been biochemically and biophysically well characterized (Golan, 1989;Bennett, 1990; Bennett and Gilligan, 1993; Mohandas and Evans, 1994).The results obtained by these studies support a “membrane skeleton fence model” in which the cytoplasmic portion of a membrane protein collides with the membrane skeletal “meshwork” because of steric hindrance, thus resulting in the temporal confinement of the protein within the mesh (compartment, Figure 1A). Membrane proteins escape from these compartments and hop to adjacent ones because of the dynamic properties of the membrane skeleton. We predict that time-dependent fluctuations in the distance between the membrane and the skeleton are large or the membrane–skeleton network connections form and break continuously, as expected in a chemical equilibrium, providing membrane proteins with the opportunity to pass through the mesh barrier. In the case of band 3, macroscopic diffusion within the erythrocyte membrane occurs as the result of a series of such intercompartmental hops.The scientific content of this work has been published previously (Tomishige et al., 1998), and the purpose of the present essay is to give the reader a glimpse of the raw data from this study, as recorded by both normal and high-speed video microscopy. We hope the video sequences attached to this article will help the viewing audience develop a feel for how band 3 interacts with the membrane skeleton and undergoes intercompartmental hop diffusion within the erythrocyte ghost membrane.VIDEO SEQUENCESVideo Sequence 1: Intercompartmental Hops of Band 3 Molecules in the Erythrocyte MembraneThe movement of band 3 in human erythrocyte ghosts in a hypotonic medium was observed at the level of a single molecule or a small number of molecules. The erythrocyte ghost membrane was stably attached to a coverslip coated with poly-l-lysine. Band 3 was labeled with 40-nm colloidal gold particles conjugated with the Fab fragments of anti-band 3 antibodies (Figure 1A). We have provided evidence that most gold particles are bound to a single band 3 molecule (Tomishige et al., 1998). The movement of gold particles attached to band 3 was observed by contrast-enhanced bright-field microscopy at 37°C, with a temporal resolution of up to 0.22 ms, using a high-speed video camera.Fig. 1. (A) The model to explain the confined diffusion of band 3 in the membrane (the membrane skeleton fence model). (B) Movement of gold particles attached to band 3 in the erythrocyte membrane, observed with a temporal resolution of 8 ms (4 times greater than the normal video rate).Video Sequence 1 shows the movement of band 3, which was recorded at a time interval of 8 ms, and is replayed at video rate (30 frames/s; see Figure 1B). Approximately two-thirds of the gold particles attached to band 3 undergo hop diffusion: as shown in Video Sequence 1, a band 3 molecule is confined within a compartment of 110 nm in diameter (on average), and on average hops to an adjacent compartment every 350 ms. This sequence contains three subsequences: the first shows a hop, the second shows two consecutive hops, and the third displays repeated hops between two domains. Band 3 molecules undergo macroscopic diffusion as the result of a series of such hops between compartments. Within a single compartment, the rate of free diffusion of band 3 molecules is only limited by the membrane viscosity, which is 5.3 × 10−9 cm2/s. The important point of this video sequence is to show that band 3 molecules do not undergo gradual movements but rather hop from one area to an adjacent area. Movements within a compartment are very small (∼3 pixels) and reflect the mesh size of the membrane-skeleton network of ∼110 nm.Video Sequence 2: Long-term Movement of Band 3: Temporal Binding of Band 3 to the Membrane SkeletonThe remaining one-third of the band 3 population exhibited random oscillatory movements within a small area of the membrane of <100 nm during the 30 s observation period. These particles do not show macroscopic diffusion and are likely to represent the band 3 molecules that are strongly bound to the membrane skeleton.Band 3 molecules that were undergoing hop diffusion eventually traversed the entire surface of the erythrocyte membrane. During observations longer than 10 min, almost all band 3 molecules were seen to attach to and detach from the skeletal network. In Video Sequence 2, band 3 sometimes stops, remains at that spot for several minutes, and then resumes hop diffusion (Figure 2; a time-lapse recording of every 250 ms, which is replayed at video rate). Such binding may be mediated through ankyrin.Fig. 2. Long-term observations of the movement of band 3 in the erythrocyte membrane. Images recorded every 250 ms using time-lapse video microscopy.Video Sequence 3: Cleavage of the Cytoplasmic Portion of Band 3 Enhances the Intercompartmental Hop RateBecause the erythrocyte membrane is compartmentalized with regard to the lateral diffusion of band 3 (Figure 1A), we next asked what is the molecular basis of the compartmentalized structure of the erythrocyte membrane. Our working hypothesis for the temporal confinement of (two-thirds of) band 3 is based on the steric hindrance of band 3 mobility imposed by the membrane skeleton; i.e., the cytoplasmic domain of band 3 collides with the membrane skeleton, which results in the temporal confinement of band 3 within the mesh of the membrane skeleton of ∼110 nm. To test this hypothesis, the cytoplasmic portion of band 3 was cleaved off by mild trypsin treatment of erythrocyte ghosts (Figure 3). Under the conditions used, most of the cytoplasmic portions of band 3 and ankyrin were cleaved, but the spectrin network (spectrin, actin, and protein 4.1) remained basically intact.Fig. 3. Movement of trypsin-cleaved band 3 was recorded with temporal resolutions of 8 and 0.89 ms.Video Sequence 3a shows the movement of the cleaved band 3 recorded every 8 ms (which is the same as that for Video Sequence 1) and is replayed at a normal video rate (thus, the time is expanded by a factor of 4.1). On this time scale, the cleaved band 3 apparently undergoes simple Brownian diffusion without hopping. The frame interval was then decreased to every 0.89 ms, and the same cleaved band 3 now shows intercompartmental hops (Video Sequence 3b; in this video clip, the time is expanded by a factor of 38). Although trypsin treatment increased the hop rate of band 3 by a factor of 6, the diffusion coefficient within the compartment and the compartment size remained the same. These results indicate an involvement of the cytoplasmic portion of band 3 in the rate of hopping across the boundaries of compartments and are consistent with the membrane skeleton fence model (Figure 1).Video Sequence 4: Deformation of the Membrane Skeletal Network Using Optical TweezersTo further observe interactions between membrane-spanning proteins and the membrane skeleton, we have developed a method to deform the membrane skeletal network using optical tweezers. Optical tweezers were used to attach a latex bead of 1 μm in diameter, coated with anti-band 3 IgG, to the center of an erythrocyte ghost membrane (Figure4). Because such a bead can simultaneously bind to many band 3 molecules, of which ∼30% are linked to the membrane skeleton, we expected that the membrane skeleton can be dragged by moving the bead by optical tweezers. Under our experimental conditions, the maximum force applied to the latex bead by our optical tweezers was ∼20 pN. To visualize the deformation of the network, 40-nm colloidal gold particles coated with anti-spectrin antibodies were attached to spectrin. In these experiments, gold particles could diffuse into the intracellular aqueous space of the ghost, because the ghosts had not been resealed in the present experiment.Fig. 4. Deformation of the membrane skeleton using optical tweezers. A 1-μm latex bead, coated with anti-band 3 IgG, bound multiple band 3 molecules, of which ∼30% are attached to the membrane skeleton. By dragging the latex bead, it was possible to deform the membrane skeleton, which can be observed by single particle tracking using gold particles specifically attached to spectrin on the cytoplasmic surface of the erythrocyte ghost membrane.Video Sequence 4 shows the movement of 40-nm gold particles bound to spectrin on the internal surface of the membrane, while the latex bead bound to the membrane skeleton was dragged by the optical trap. These image data suggest that deformation/displacements of the membrane skeleton occur even when a distant part of the membrane skeleton is being dragged with an optical trap. Note that in comparison the contour of the cell was negligibly deformed, which indicates that the dragging caused deformation of the membrane skeletal network, rather than translation of the entire membrane.Video Sequence 5: Dragging of the Membrane Skeletal Network Forced the Displacement of Unbound Band 3 Along with the Movement of the NetworkTo discover whether the deformation/displacement of the membrane skeleton network causes forced movement of band 3, band 3 (in place of spectrin) was labeled with 40-nm gold particles, and the latex bead bound to the membrane skeleton was dragged using optical tweezers. We paid attention only to band 3 undergoing hop diffusion (unbound band 3). If unbound band 3 molecules collide with the membrane skeleton, they will be carried along with the movement of the membrane skeleton meshes (Figure 5A). If they do not collide with the membrane skeleton, their movement will not be affected by deformation/displacements of the membrane skeleton.Fig. 5. (A) Effect of lateral dragging of a 1-μm latex bead attached to the membrane skeleton on the movement of several band 3 molecules capable of undergoing hop diffusion. (B) The gold particles bound to band 3 undergoing hop diffusion followed the bead when it was dragged toward the left at a rate of 0.15 μm/s.In Video Sequence 5a, the large bead in the center (attached to the membrane skeleton via band 3 molecules) was dragged toward the left, while, at the same time, the movement of band 3-gold that had previously been undergoing hop diffusion was observed. The sequence is shown in real time (video rate recording). When the latex bead was dragged toward the left at a rate of 1.8 μm/s, the gold particles attached to band 3 (seen on the right) were displaced in the direction of dragging of the latex bead. When dragging of the latex bead was stopped, the same band 3 molecules continued a hop diffusion, indicating that they are not bound to the membrane skeleton. When the large bead was moved back toward its original position, the gold-bound band 3 molecules returned, but not to their original positions, because of the hop diffusion that kept occurring during this sequence. This result strongly suggests that the cytoplasmic domain of band 3 collides with the membrane skeleton.A possible explanation for the forced movement of band 3 by dragging the membrane skeleton may be a hydrodynamic coupling effect, because all membrane proteins and lipids that are bound to the membrane skeleton network will move in concert with the membrane skeleton. We examined the extent of this effect by lowering the rate of dragging of the membrane skeleton (by a factor of 12, to 0.15 μm/s; Video Sequence 5b). Even at this slow rate of dragging, the gold particles attached to band 3 still followed the large bead’s movements (Figure 5B). These results show that the band 3 molecules undergoing hop diffusion do indeed collide with the membrane skeleton network, and this results in their temporal confinement within a compartment of the membrane skeleton.Video Sequence 6: Dragging of the Membrane Skeleton Does Not Induce Displacement of Phospholipids Located on the Outer SurfaceTo further examine the possibility that the hydrodynamic coupling was responsible for the concerted movement of band 3 as the membrane skeleton was dragged, we next examined the effect of dragging the membrane skeleton on the lipids in the outer leaflet of the membrane. Direct interaction of these lipids with the membrane skeleton is not possible. As such, any effect must be caused by hydrodynamic coupling through the fluid bilayer. Fluorescein-phosphatidylethanolamine was artificially incorporated into the erythrocyte ghost and labeled with gold particles coated with anti-fluorescein Fab.In Video Sequence 6 (Figure 6), a gold particle bound to fluorescein-phosphatidylethanolamine in the outer leaflet of the membrane is seen near the right edge of the cell. This particle is seen to undergo rapid Brownian diffusion. The membrane skeleton was then dragged toward the left at a rate of 1.8 μm/s; however, the lipid showed no sign of following the large bead and thus the membrane skeleton.Fig. 6. Effect of lateral dragging of a 1-μm latex bead attached to the membrane skeleton on the movement of a lipid that is located in the outer leaflet of the cell membrane.CONCLUSIONThe image data shown in this video essay indicate that the erythrocyte membrane is compartmentalized with regard to the lateral diffusion of band 3 and presumably other transmembrane proteins. The confined diffusion is most likely caused by the collision of the cytoplasmic portion of band 3 with the membrane skeleton network (membrane–skeleton fence model). These conclusions could only be reached by studying the movements of single band 3 molecules using single particle tracking techniques and by direct mechanical manipulations of the membrane skeleton using optical tweezers. Because single molecule techniques can be applied to study the movements of various other proteins in the supramolecular complexes contained in living cells, they provide powerful tools for cell biologists to investigate the dynamics and underlying mechanisms for many cellular processes.ACKNOWLEDGMENTSWe thank Professor Gerard Marriott and Dr. Kenneth Ritchie for critical reading of this manuscript and Prof. Yuichi Takakuwa for the gift of antibodies.REFERENCES1 Bennett V. (1990). Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm.. Physiol. Rev. 70, 1029-1065. Crossref, Medline, Google Scholar2 Bennett V., Gilligan D.M. (1993). The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane.. Annu. Rev. Cell Biol. 9, 27-66. Crossref, Medline, Google Scholar3 Edidin M., Kuo S.C., Sheetz M.P. (1991). Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers.. Science 254, 1379-1382. Crossref, Medline, Google Scholar4 Golan D.E. (1989). Red blood cell membrane protein and lipid diffusion. In: Red Blood Cell Membranes, Ed. P. AgreJ.C. Parker, New York: Marcel Dekker, 367-400. Google Scholar5 Jacobson K., Sheets E.D., Simson R. (1995). Revisiting the fluid mosaic model of membranes.. Science 268, 1441-1442. Crossref, Medline, Google Scholar6 Kusumi A., Sako Y. (1996). Cell surface organization by the membrane skeleton.. Curr. Opin. Cell Biol. 8, 566-574. Crossref, Medline, Google Scholar7 Kusumi A., Sako Y., Fujiwara T., Tomishige M. (1998). Application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins.. Methods Cell Biol. 55, 173-194. Crossref, Medline, Google Scholar8 Kusumi A., Sako Y., Yamamoto M. (1993). Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.. Biophys. J. 65, 2021-2040. Crossref, Medline, Google Scholar9 Luna E.J., Hitt A.L. (1992). Cytoskeleton-plasma membrane interactions.. Science 258, 955-964. Crossref, Medline, Google Scholar10 Mohandas N., Evans E. (1994). Mechanical properties of the red cell membrane in relation to molecular structure and genetic defects.. Annu. Rev. Biophys. Biomol. Struct. 23, 787-818. Crossref, Medline, Google Scholar11 Sako Y., Kusumi A. (1994). Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis.. J. Cell Biol. 125, 1251-1264. Crossref, Medline, Google Scholar12 Sako Y., Kusumi A. (1995). Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether.. J. Cell Biol. 129, 1559-1574. Crossref, Medline, Google Scholar13 Sako Y., Nagafuchi A., Tsukita S., Takeichi M., Kusumi A. (1998). Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.. J. Cell Biol. 140, 1227-1240. Crossref, Medline, Google Scholar14 Saxton M.J., Jacobson K. (1997). Single-particle tracking: applications to membrane dynamics.. Annu. Rev. Biophys. Biomol. Struct. 26, 373-399. Crossref, Medline, Google Scholar15 Sheets E.D., Lee G.M., Simson R., Jacobson K. (1997). Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane.. Biochemistry 36, 12449-12458. Crossref, Medline, Google Scholar16 Sheets E.D., Simson R., Jacobson K. (1995). New insights into membrane dynamics from the analysis of cell surface interactions by physical methods.. Curr. Opin. Cell Biol. 7, 707-714. Crossref, Medline, Google Scholar17 Simson R., Yang B., Moore S.E., Doherty P., Walsh F.S., Jacobson K.A. (1998). Structural mosaicism on the submicron scale in the plasma membrane.. Biophys. J. 74, 297-308. Crossref, Medline, Google Scholar18 Tomishige M., Sako Y., Kusumi A. (1998). Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.. J. Cell Biol. 142, 989-1000. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited byBibliographySeptin Interferes with the Temperature-Dependent Domain Formation and Disappearance of Lipid Bilayer Membranes15 November 2016 | Langmuir, Vol. 32, No. 48Nanoscopic compartmentalization of membrane protein motion at the axon initial segment3 October 2016 | Journal of Cell Biology, Vol. 215, No. 1Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes6 September 2016 | PLOS ONE, Vol. 11, No. 9Modeling of band-3 protein diffusion in the normal and defective red blood cell membrane1 January 2016 | Soft Matter, Vol. 12, No. 15Crowding, Diffusion, and Biochemical ReactionsReceptor Displacement in the Cell Membrane by Hydrodynamic Force Amplification through NanoparticlesBiophysical Journal, Vol. 105, No. 1Force probing of individual molecules inside the living cell is now a reality17 October 2012 | Nature Chemical Biology, Vol. 8, No. 11Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine ErythrocytesJournal of Biological Chemistry, Vol. 287, No. 6Cytoskeleton-Induced Mesoscale Domains31 May 2011Analysis of the kinetics of band 3 diffusion in human erythroblasts during assembly of the erythrocyte membrane skeleton18 August 2010 | British Journal of Haematology, Vol. 150, No. 5Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy20 February 2010 | FEBS Letters, Vol. 584, No. 9ReferencesImaging of the diffusion of single band 3 molecules on normal and mutant erythrocytesBlood, Vol. 113, No. 24Diffusion in a Fluid Membrane with a Flexible Cortical CytoskeletonBiophysical Journal, Vol. 96, No. 3(Un)Confined Diffusion of CD59 in the Plasma Membrane Determined by High-Resolution Single Molecule MicroscopyBiophysical Journal, Vol. 92, No. 10Neural membrane microdomains as computational systems: Toward molecular modeling in the study of neural diseaseBiosystems, Vol. 87, No. 1Thermo-elasticity and adhesion as regulators of cell membrane architecture and function26 October 2006 | Journal of Physics: Condensed Matter, Vol. 18, No. 45Neural membrane field effects in a cytoskeleton corral: Microdomain regulation of impulse propagation1 January 2004 | International Journal of Quantum Chemistry, Vol. 100, No. 6The membrane lateral domain approach in the studies of lipid–protein interaction of GPI-anchored bovine erythrocyte acetylcholinesterase6 July 2004 | European Biophysics Journal, Vol. 33, No. 8Heterogeneous Motion of Secretory Vesicles in the Actin Cortex of Live Cells: 3D Tracking to 5-nm Accuracy7 October 2004 | The Journal of Physical Chemistry A, Vol. 108, No. 45LDL transcytosis by protein membrane diffusionThe International Journal of Biochemistry & Cell Biology, Vol. 36, No. 3Soft Picture of Lateral Heterogeneity in BiomembranesJournal of Membrane Biology, Vol. 196, No. 2Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization22 June 2003 | Nature Cell Biology, Vol. 5, No. 7Observing Membrane Protein Diffusion at Subnanometer ResolutionJournal of Molecular Biology, Vol. 327, No. 5Phospholipids undergo hop diffusion in compartmentalized cell membrane10 June 2002 | Journal of Cell Biology, Vol. 157, No. 6Journal of Magnetic Resonance, Vol. 157, No. 1Cellular Membranes Studied by Photonic Force MicroscopyDeformation-Enhanced Fluctuations in the Red Cell Skeleton with Theoretical Relations to Elasticity, Connectivity, and Spectrin UnfoldingBiophysical Journal, Vol. 81, No. 6Single Molecule Imaging of Green Fluorescent Proteins in Living Cells: E-Cadherin Forms Oligomers on the Free Cell SurfaceBiophysical Journal, Vol. 80, No. 6 Vol. 10, No. 8 August 01, 19992475-2802 Supplemental MaterialsMetrics Downloads & Citations Downloads: 61Citations: 30 History Submitted: 18 March 1999 Accepted: 7 June 1999 InformationCopyright © 1999 The American Society for Cell BiologyACKNOWLEDGMENTSWe thank Professor Gerard Marriott and Dr. Kenneth Ritchie for critical reading of this manuscript and Prof. Yuichi Takakuwa for the gift of antibodies.PDF download