Chapter 1 - Tubular liposomes with variable permeability for reconstitution of FtsZ rings.
نویسندگان
چکیده
We have developed a system for producing tubular multilamellar liposomes that incorporate the protein FtsZ on the inside. We start with a mixture of spherical multilamellar liposomes with FtsZ initially on the outside. Shearing forces generated by applying a coverslip most likely distort some of the spherical liposomes into a tubular shape, and causes some to leak and incorporate FtsZ inside. We describe protocols for liposome preparation, and for preparing membrane-targeted FtsZ that can assemble contractile Z rings inside the tubular liposomes. We also describe the characterization of the multilamellar liposomes in terms of the permeability or leakiness for a small fluorescent dye and larger protein molecules. These liposomes may be useful for reconstitution of other biological systems.
منابع مشابه
Inside-out Z rings--constriction with and without GTP hydrolysis.
The bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opp...
متن کاملNegative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.
FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ ring...
متن کاملLiposome division by a simple bacterial division machinery.
We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed ...
متن کاملFtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one.
FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator....
متن کاملCurved FtsZ protofilaments generate bending forces on liposome membranes.
We have created FtsZ-YFP-mts where an amphipathic helix on the C-terminus tethers FtsZ to the membrane. When incorporated inside multi-lamellar tubular liposomes, FtsZ-YFP-mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ-YFP-mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposom...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Methods in enzymology
دوره 464 شماره
صفحات -
تاریخ انتشار 2009