Microtubules associate with actin-containing filaments at discrete sites along the ventral surface of Allogromia reticulopods

We have investigated the distribution of actin and microtubules in pseudopodial networks (reticulopods) of the protozoan Allogromia sp., strain NF, in order to help elucidate the respective roles these components play in network organization and motility. Double-label fluorescence studies with tubulin antibodies and tetramethyl-rhodamine (TMR)-phalloidin reveal that microtubules and filamentous actin co-localize in regions where trunk pseudopods contact the substratum and splay to form the pseudopodial network; distal to these regions the network contains numerous microtubules but little or no F-actin. Similar results were obtained using various commercial actin antibodies. Correlative anti-actin immunofluorescence and high-voltage electron microscopy of serial 0.25 micron sections reveal that actin is contained within discrete electron-opaque, fan-shaped structures distributed along the cytoplasmic aspect of the ventral reticulopodial membrane. Electron microscopy of serial 100 nm sections from conventionally fixed specimens confirms that these actin-rich plaques are composed of a felt of roughly parallel, 5 nm diameter filaments. A subset of parallel and often bundled microtubules is enmeshed within, or contacts the periphery of, these filament plaques. Upon leaving a plaque, bundled microtubules frequently splay into smaller bundles. These observations are consistent with the hypothesis that interactions between microtubules and actin-containing microfilaments, particularly at substratum adhesion points, are involved in various aspects of reticulopodial motility, particularly network morphogenesis and cell body locomotion.