Limits...
Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

Fujiwara TK, Iwasawa K, Kalay Z, Tsunoyama TA, Watanabe Y, Umemura YM, Murakoshi H, Suzuki KG, Nemoto YL, Morone N, Kusumi A - Mol. Biol. Cell (2016)

Bottom Line: Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion.The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion.These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

View Article: PubMed Central - PubMed

Affiliation: Center for Meso-Bio Single-Molecule Imaging, Institute for Integrated Cell-Material Sciences, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus

The MSK fence and anchored-TM-protein picket model, and the single-molecule tracking methods used in this study. (A) Fence-and-pickets model. The PM can be partitioned into compartments, and both TM proteins and lipids undergo short-term confined diffusion within a compartment and long-term hop movements between these compartments, which is termed hop diffusion. Temporary confinement within the compartment is induced by the actin-MSK “fences” and the anchored-TM-protein “pickets” anchored to and aligned along the actin MSK. In this study, we examined the movements of DOPE and TfR (a native dimer). Side view, a variety of TM proteins (temporarily) bind to the MSK, and these MSK-anchored TM proteins act like “pickets.” Bottom view, the PM cytoplasmic surface, viewed from inside the cell, showing the MSK “fence” model. Top view, many TM proteins are (temporarily) anchored to and aligned along the actin MSK, exerting hydrodynamic circumferential-slowing (enhanced viscosity) and steric-hindrance effects on PM molecules that approach the anchored TM-proteins. (B) Experimental design for SFMT and SPT. For SFMT, TfR tagged with Cy3-Tf (a) and Cy3-DOPE (b) were used. For SPT, TfR tagged with 40-nm-diameter colloidal gold particles coated with a small number of transferrin molecules were used (c). For colloidal-gold labeling of DOPE (d), gold probes coated with anti-fluorescein antibody Fab fragments were bound to fluorescein-conjugated DOPE, which was preincorporated in the PM. The fluorescein moiety was used as a tag for the antibody Fab rather than a fluorescent probe. (C) Images of Cy3 and colloidal-gold probes and their trajectories at video rate for 3 s, observed on the top surface of PtK2 cells. Here a–d are the same as in B.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4814218&req=5

Figure 1: The MSK fence and anchored-TM-protein picket model, and the single-molecule tracking methods used in this study. (A) Fence-and-pickets model. The PM can be partitioned into compartments, and both TM proteins and lipids undergo short-term confined diffusion within a compartment and long-term hop movements between these compartments, which is termed hop diffusion. Temporary confinement within the compartment is induced by the actin-MSK “fences” and the anchored-TM-protein “pickets” anchored to and aligned along the actin MSK. In this study, we examined the movements of DOPE and TfR (a native dimer). Side view, a variety of TM proteins (temporarily) bind to the MSK, and these MSK-anchored TM proteins act like “pickets.” Bottom view, the PM cytoplasmic surface, viewed from inside the cell, showing the MSK “fence” model. Top view, many TM proteins are (temporarily) anchored to and aligned along the actin MSK, exerting hydrodynamic circumferential-slowing (enhanced viscosity) and steric-hindrance effects on PM molecules that approach the anchored TM-proteins. (B) Experimental design for SFMT and SPT. For SFMT, TfR tagged with Cy3-Tf (a) and Cy3-DOPE (b) were used. For SPT, TfR tagged with 40-nm-diameter colloidal gold particles coated with a small number of transferrin molecules were used (c). For colloidal-gold labeling of DOPE (d), gold probes coated with anti-fluorescein antibody Fab fragments were bound to fluorescein-conjugated DOPE, which was preincorporated in the PM. The fluorescein moiety was used as a tag for the antibody Fab rather than a fluorescent probe. (C) Images of Cy3 and colloidal-gold probes and their trajectories at video rate for 3 s, observed on the top surface of PtK2 cells. Here a–d are the same as in B.

Mentions: One of the most controversial issues about the domain structure of the plasma membrane is the actin-induced compartmentalization of the PM. Transmembrane (TM) proteins, phospholipids, and glycosylphosphatidylinositol (GPI)-anchored proteins have been proposed to undergo short-term confined diffusion within compartments of ∼100 nm formed by the actin-filament meshwork bound to the PM inner surface (Morone et al., 2006), called the membrane-skeleton (MSK) fence, and various TM proteins lining the actin-based MSK, called anchored TM-protein pickets (Figure 1A; Kusumi et al., 2005, 2012; Kalay et al., 2014). Of importance, these pickets exert not only steric hindrance effects but also hydrodynamic friction effects on the surrounding molecules, thus inducing confining effects on membrane molecules. Membrane molecules undergo long-term hop movements between compartments, and the long-term macroscopic diffusion coefficients are reduced by a factor of ∼20 from the microscopic diffusion coefficient within a compartment, which is comparable to the single diffusion coefficients found in the blebbed PM, where actin filaments are largely depleted, and in artificial lipid membranes (6–10 μm2/s; Fujiwara et al., 2002; Kusumi et al., 2012).


Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

Fujiwara TK, Iwasawa K, Kalay Z, Tsunoyama TA, Watanabe Y, Umemura YM, Murakoshi H, Suzuki KG, Nemoto YL, Morone N, Kusumi A - Mol. Biol. Cell (2016)

The MSK fence and anchored-TM-protein picket model, and the single-molecule tracking methods used in this study. (A) Fence-and-pickets model. The PM can be partitioned into compartments, and both TM proteins and lipids undergo short-term confined diffusion within a compartment and long-term hop movements between these compartments, which is termed hop diffusion. Temporary confinement within the compartment is induced by the actin-MSK “fences” and the anchored-TM-protein “pickets” anchored to and aligned along the actin MSK. In this study, we examined the movements of DOPE and TfR (a native dimer). Side view, a variety of TM proteins (temporarily) bind to the MSK, and these MSK-anchored TM proteins act like “pickets.” Bottom view, the PM cytoplasmic surface, viewed from inside the cell, showing the MSK “fence” model. Top view, many TM proteins are (temporarily) anchored to and aligned along the actin MSK, exerting hydrodynamic circumferential-slowing (enhanced viscosity) and steric-hindrance effects on PM molecules that approach the anchored TM-proteins. (B) Experimental design for SFMT and SPT. For SFMT, TfR tagged with Cy3-Tf (a) and Cy3-DOPE (b) were used. For SPT, TfR tagged with 40-nm-diameter colloidal gold particles coated with a small number of transferrin molecules were used (c). For colloidal-gold labeling of DOPE (d), gold probes coated with anti-fluorescein antibody Fab fragments were bound to fluorescein-conjugated DOPE, which was preincorporated in the PM. The fluorescein moiety was used as a tag for the antibody Fab rather than a fluorescent probe. (C) Images of Cy3 and colloidal-gold probes and their trajectories at video rate for 3 s, observed on the top surface of PtK2 cells. Here a–d are the same as in B.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814218&req=5

Figure 1: The MSK fence and anchored-TM-protein picket model, and the single-molecule tracking methods used in this study. (A) Fence-and-pickets model. The PM can be partitioned into compartments, and both TM proteins and lipids undergo short-term confined diffusion within a compartment and long-term hop movements between these compartments, which is termed hop diffusion. Temporary confinement within the compartment is induced by the actin-MSK “fences” and the anchored-TM-protein “pickets” anchored to and aligned along the actin MSK. In this study, we examined the movements of DOPE and TfR (a native dimer). Side view, a variety of TM proteins (temporarily) bind to the MSK, and these MSK-anchored TM proteins act like “pickets.” Bottom view, the PM cytoplasmic surface, viewed from inside the cell, showing the MSK “fence” model. Top view, many TM proteins are (temporarily) anchored to and aligned along the actin MSK, exerting hydrodynamic circumferential-slowing (enhanced viscosity) and steric-hindrance effects on PM molecules that approach the anchored TM-proteins. (B) Experimental design for SFMT and SPT. For SFMT, TfR tagged with Cy3-Tf (a) and Cy3-DOPE (b) were used. For SPT, TfR tagged with 40-nm-diameter colloidal gold particles coated with a small number of transferrin molecules were used (c). For colloidal-gold labeling of DOPE (d), gold probes coated with anti-fluorescein antibody Fab fragments were bound to fluorescein-conjugated DOPE, which was preincorporated in the PM. The fluorescein moiety was used as a tag for the antibody Fab rather than a fluorescent probe. (C) Images of Cy3 and colloidal-gold probes and their trajectories at video rate for 3 s, observed on the top surface of PtK2 cells. Here a–d are the same as in B.
Mentions: One of the most controversial issues about the domain structure of the plasma membrane is the actin-induced compartmentalization of the PM. Transmembrane (TM) proteins, phospholipids, and glycosylphosphatidylinositol (GPI)-anchored proteins have been proposed to undergo short-term confined diffusion within compartments of ∼100 nm formed by the actin-filament meshwork bound to the PM inner surface (Morone et al., 2006), called the membrane-skeleton (MSK) fence, and various TM proteins lining the actin-based MSK, called anchored TM-protein pickets (Figure 1A; Kusumi et al., 2005, 2012; Kalay et al., 2014). Of importance, these pickets exert not only steric hindrance effects but also hydrodynamic friction effects on the surrounding molecules, thus inducing confining effects on membrane molecules. Membrane molecules undergo long-term hop movements between compartments, and the long-term macroscopic diffusion coefficients are reduced by a factor of ∼20 from the microscopic diffusion coefficient within a compartment, which is comparable to the single diffusion coefficients found in the blebbed PM, where actin filaments are largely depleted, and in artificial lipid membranes (6–10 μm2/s; Fujiwara et al., 2002; Kusumi et al., 2012).

Bottom Line: Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion.The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion.These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

View Article: PubMed Central - PubMed

Affiliation: Center for Meso-Bio Single-Molecule Imaging, Institute for Integrated Cell-Material Sciences, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus