Endosomal sorting complex required for transport III (ESCRT-III) proteins function in AMD 070 multivesicular body biogenesis and viral budding. 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. Introduction Multivesicular bodies (MVBs) are mid-stage endosomes that contain intraluminal vesicles (ILVs). ILVs are generated by invagination and scission from the limiting membrane of the endosome. Ultimately most ILVs are delivered to lysosomes enabling degradation of transmembrane proteins and lipids (Katzmann et al. 2002 Gruenberg and Stenmark 2004 for reviews see Babst 2005 Hurley and Emr 2006 However in certain specialized cells MVBs also fuse with the plasma membrane to secrete their ILVs as AMD 070 entities called exosomes which subserve a variety of important functions in the immune system and elsewhere (Stoorvogel et al. 2002 Thery et al. 2002 Fevrier and Raposo 2004 A great deal of attention has recently focused on understanding how proteins are sorted into MVBs and how ILVs actually form. Among the proteins involved in creating ILVs are at least 18 that were identified via genetic studies of vacuolar protein sorting in the yeast suggest that PTP2C they act late in the process after ESCRT-I and ESCRT-II. Missing from current models is an understanding of which factors drive the required membrane deformation and eventual separation of ILVs from the limiting membrane of the endosome (for reviews see Hurley and Emr 2006 Williams and Urbe 2007 In the present study we find that the ESCRT-III proteins hSnf7-1/CHMP4A and hSnf7-2/CHMP4B assemble into circular membrane-associated polymers that can be engaged to deform the membrane to which they are attached. We propose that membrane-associated ESCRT-III polymers similar to these may contribute to delineating and generating vesicles within the lumen of the MVB. Results hSnf7 assembles into homopolymeric filaments on the membrane To study the organization of ESCRT-III-containing polymers by quick-freeze deep-etch EM (DEEM) we took advantage of our earlier observation that overexpressed hSnf7 (CHMP4) protein accumulate in areas on or next to the plasma membrane aswell as on inner mainly endosomal compartments (Lin et al. 2005 Shim et al. 2007 The plasma membrane can be more available to DEEM than inner organelles because cells want only become “unroofed” to acquire expansive images from it and constructions mounted on it (Heuser 2000 b). The anaglyph in the very best of Fig. 1 displays a typical picture of the internal surface AMD 070 from the plasma membrane of the COS-7 cell which in cases like this can be transiently transfected having a plasmid encoding full-length FLAG-tagged hSnf7-1 (CHMP4A). Noticeable for the membrane will be the typical cortical cytoskeletal parts including actin filaments and polygonal clathrin lattices but additionally you can find abundant filaments that are curved and interconnected to create a number of round arrays. In a few areas these fresh filaments intermingle with AMD 070 actin and clathrin whereas in the areas they may be therefore abundant that they displace these regular residents of the plasma membrane. It is important to note that transiently transfected COS-7 cells produce ESCRT-III proteins at levels that greatly exceed the concentration of their endogenous counterparts (unpublished data) creating a situation in which we can study the behavior of transfected proteins without significant contributions from endogenous proteins or binding partners. 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 … Higher magnification DEEM views of cells expressing hSnf7-1 or the related hSnf7-2 (CHMP4B) show that the novel filaments have a unit diameter of ～5-6 nm (including the ～2-nm thickness of the platinum replica that coats them) and appear to be tightly associated with the plasma membrane (Fig. 1.