THE MEMBRANE SKELETON OF DICTYOSTELIUM AMOEBAE (CELL MOTILITY, SPECTRIN, CYTOSKELETON, MICROFILAMENT-MEMBRANE INTERACTIONS, CAPPING)
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I used morphological, immunocytochemical, and biochemical techniques to study the organization of the membrane skeleton, the fine structure of the cortex, and a potential microfilament-membrane linking protein in amoebae of Dictyostelium discoideum.;Amoebae have a random shape. Upon Concanavalin A (Con A) capping, they become polarized with a cap at one end and a pseudopod at the opposite end. Unchallenged cells have a random distribution of intramembrane particles (IMPs) while capped cells have a gradient in the numerical density of IMPs with the highest concentration of IMPs at the cap and the lowest concentration at the pseudopod. Differences in density between the cap and other regions of the cell is two- to threefold for all IMPs, but can be as much as sevenfold for >12 nm IMPs. Development of cell polarity during capping may be due to the asymmetric distribution of IMPs, which may cause asymmetric ion currents across cells.;Fine structure of microfilament-membrane contacts can be examined in isolated cortices. Inclusion of EGTA and KPO(,4) during cell lysis and addition of phalloidin were crucial to isolating stable cortices. Meshworks of microfilaments contact cell membranes at their barbed ends. Lateral contacts mediated by short, rod shaped bridges (length = 15nm) bind along microfilaments with a periodicity of 36nm. There are approximately 25,000 microfilament-membrane contacts per isolated cortex. Cortices incubated with S-1 sufficient to saturate binding sites on microfilaments have a threefold loss in the number of contacts. Therefore, caution must be used when information about precise location of microfilaments and contacts to cell membrane is required.;Amoebae and cortices contain immunoreactive analogues of chicken brain fodrin (Mr 220K, 70K). 220K, extracted from cortices with 0.2 M NaCl and 10 mM MgATP and purified by gel filtration and sucrose density gradient sedimentation, is a rod shaped protein (length = 118nm, S value = 9.3, Stokes' radius = 13.5nm) which binds laterally to F-actin. The immunoreactive analogue, closely associated with microfilaments and cell membrane, co-patches and co-caps with Con A. Isolation of a fodrin-like protein from an amoeboid cell opens the way for analysis of the regulation of actin-membrane interactions.
- Theses and Dissertations