The applicant has purified a high molecular weight actin binding protein (designated HMWP) from HeLa cells. HMWP is homologous to the filamins and, therefore, may help establish cytoarchitecture in HeLa cells by cross-linking actin filaments in vivo. In situ localization of filamins using electron microscopy has not yet been reported and, therefore, we shall explore the intracellular localization of HMWP using immunocytochemistry at the electron microscopic level. This will allow us to determine if HMWP is actually concentrated in cortical cytoplasm (suggested by our immunofluorescence studies), to study the distribution of HMWP relative to the dense regions of the stress fibers, and to confirm that HMWP is present in stress fibers but absent from microvilli (suggested by our immunofluorescence studies). To study how intrinsic properties of the interaction of HMWP with actin might influence its in situ localization, we shall determine the polarity of the actin filaments in the bundles induced in vitro by HMWP. The observation or random polarity would suggest that HMWP should be present in situ only in structures with a similar organization, such as stress fibers; the observation of a single polarity would suggest that it should also be present in situ in structures containing actin filaments of 1 polarity, such as the microvillus and, therefore that the random polarity of the filaments in stress fibers might be caused by interdigitation of bundles of opposite intrinsic polarity. The applicant recently observed incorporation of HMWP into striated paracrystals in vitro. To determine if this is a general property of the filamins, we shall investigate smooth muscle filamin for the ability to form paracrystals. If it does not, then we shall investigate HeLa cells for the presence of factor(s) that induce incorporation of HMWP as well as filamin into paracrystals. We shall use electron microscopy to investigate how HMWP is incorporated into paracrystals. We shall examine HMWP and paracrystals after rotary shadowing, thin sections of paracrystals, and negatively stained paracrystals after decoration with antibody to HMWP tagged with colloidal gold or ferritin and after controlled proteolysis. These studies will test the hypothesis that HMWP is incorporated into paracrystals by end-to-end association of HMWP molecules and permit mapping of the domain(s) that cause incorporation into paracrystals relative to the actin binding and the dimerization domains that have previously been recognized on other filamins.
| Weihing, R R (1988) Actin-binding and dimerization domains of HeLa cell filamin. Biochemistry 27:1865-9 |
