Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

Abstract

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used "deep-etch" electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis-deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.

Figure 1.

hSnf7 proteins form curved filaments on the plasma membrane. Shown in 3D are anaglyphs of the inside of the plasma membrane of COS-7 cells expressing the constructs indicated. Use view glasses for the 3D structure (left = red). (A) Plasma membrane of cell expressing FLAG hSnf7-1. (B) Higher magnification views of membrane coated with filaments of FLAG hSnf7-1 (left), FLAG hSnf7-2 (center), and FLAG hSnf7-1 (right). Bars, 100 nm. (C) High magnification views of FLAG hSnf7-1 filaments. (D) Clathrin lattice (a), two additional panels of FLAG hSnf7-1 filaments (b and c), and three views of actin filaments (d, e, and f). Filaments in C and D have been highlighted for clarity. Panels in C and D are each 100 nm across.

Figure 2.

Filaments containing hSnf7-1–GFP show their GFP. Adding GFP to the C terminus of hSnf7-1 (hSnf7-1–GFP) creates bumpy, tightly wound filaments on the inner surface of the plasma membrane. Bar, 100 nm.

Figure 3.

hSnf7 filaments on the top surface of the cell. (top) Patterns created by hSnf7-1 filaments on the outer surface of whole cells. Shown is the top surface of a COS-7 cell transfected with FLAG hSnf7-1, fixed, and replicated without disruption. Note the subtle circular patterning of particles within the membrane. (middle) Views of hSnf7-1 filaments in the subplasmalemmal “membrane skeleton” revealed by extracting fixed whole cells with detergent. The cell on the left expresses FLAG hSnf7-1, whereas the one on the right does not. (bottom left) Fixed and extracted cell expressing higher levels of FLAG hSnf7-1. (bottom right) Fixed and extracted cell expressing hSnf7-1–mGFP. Bars, 100 nm.

Figure 4.

hSnf7/CHMP4 filaments bind VPS4B(E235Q). Anaglyphs of plasma membranes from COS cells expressing FLAG hSnf7-1 and VPS4B(E235Q)-GFP. (top) Immunodecoration with antibodies against FLAG tag on FLAG hSnf7-1 (left) and GFP in VPS4B(E235Q)-GFP (right). Yellow circles have been superimposed on the 18-nm gold particles for clarity. Note that gold particles obscure individual VPS4B particles only in the right panel. (middle) Circles of hSnf7-1 filaments with an increasing number of VPS4B(E235Q)-GFP particles bound. (bottom) Low magnification survey view of COS-7 cell plasma membrane showing hSnf7-1 arrays heavily decorated with VPS4B(E235Q)-GFP. Note that all three have small central holes. Bars, 100 nm.

Figure 5.

Buds and tubules protrude from the top surface of cells coexpressing hSnf7-1 and VPS4B(E235Q)-GFP. (top) Overview of fixed whole cell. (bottom) Higher magnification views of selected buds and tubules showing the range of observed structures. Bars, 100 nm.

Figure 6.

Protein scaffolds line buds and tubules in cells coexpressing hSnf7-1 and VPS4B(E235Q)-GFP. Fixed whole cells extracted with detergents after fixation show submembranous skeleton. (top) Uniform budlike structures on region of a cell. (bottom) Buds and tubules of varying lengths along the surface of another cell. Bars, 100 nm.

Figure 7.

Spontaneous tears along the top surface of fixed whole cells reveal the fine structure of the underlying membrane skeleton (with no detergent treatment). (top) Survey view. (bottom) Higher magnification views. Bars, 100 nm.

Figure 8.

hSnf7-1 N-terminal fragment (1–116) drives formation of everting buds. (left) Top surface of a fixed whole cell expressing hSnf7-1(1–116). (top right) Filaments and large particles on the inner surface of the plasma membrane of cells expressing hSnf7-1(1–116). (right, middle) Freeze fracture image of membrane from cells expressing hSnf7-1(1–116) grown on sapphire. (bottom right) Selected views from fixed whole cells extracted with detergents after fixation. Bars, 100 nm.

Figure 9.

VPS4B(E235Q)-GFP forms large corral-like rings on the plasma membrane of cells expressing only endogenous ESCRT-III proteins. (top) Overview of plasma membrane from a cell expressing only VPS4B(E235Q)-GFP. A GFP tag on VPS4 was used to selectively immunodecorate these particles with 18 nm gold. (bottom) Selected higher magnification views of rings formed by VPS4B(E235Q)-GFP. Bars: (top) 500 nm; (bottom) 100 nm.