Microtubules associate with actin-containing filaments at discrete sites along the ventral surface of Allogromia reticulopods SAMUEL S. BOWSER 1 *, JEFFREY L. TRAVIS 2 and CONLY L. RIEDER 1 ' 3 1 Wadstuo it h Center fur Laboratories and Research, .\'etv York State Department of Health, Albany, AT 12201, ISA ^Department of Biology, Vassal College, Poughkeepsie, AT 12601, USA ^School of Public Health, Stale University of.\'ew York, Albany, AT 12201, USA • Autho r for correspondence Summary We have investigated the distribution of actin and microtubules in pseudopodial networks (reticulo- pods) 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-rhoda- mine (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 nu- merous microtubules but little or no F-actin. Similar results -were obtained using various com- mercial actin antibodies. Correlative anti-actin immunofluorescence and high-voltage electron microscopy of serial 0-25 jum sections reveal that actin is contained within discrete electron- opaque, fan-shaped structures distributed along the cytoplasmic aspect of the ventral reticulopo- dial membrane. Electron microscopy of serial 100 nm sections from conventionally fixed speci- mens confirms that these actin-rich plaques are composed of a felt of roughly parallel, 5nm 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 ob- servations are consistent with the hypothesis that interactions between microtubules and actin- containing microfilaments, particularly at sub- stratum adhesion points, are involved in various aspects of reticulopodial motility, particularly network morphogenesis and cell body loco- motion. Key words: microfilaments, microtubules, actin, Allogromia, cell-substrate interactions. Introduction Interactions between filamentous actin (F-actin or microfilaments; MFs) and microtubules (MTs) have been demonstrated structurally and biochemically in a variety of cells (reviewed by Pollard et al. 1984). These interactions, which in vitro appear to be mediated by MT-associated proteins (MAPs), have been proposed to provide structural integrity to the cytoplasmic matrix and to participate indirectly (via the mech- anoenzyme myosin) in MT-dependent transport (Ochs, 1972; Griffith & Pollard, 1978; Sattilaro & Dentler, 1982; Hayden et al. 1983). However, at Journal of Cell Science 89, 297-307 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 present the role(s) such interactions play in cell func- tion remains speculative due, in part, to a paucity of well-defined biological systems with which to study such phenomena in vivo. The pseudopodial network (reticulopodium) of the marine foraminiferan Allogromia has proven to be an excellent model system for studying various aspects of MT function. In this protist MTs, singly or in small bundles, can be observed in flattened pseudopods with unsurpassed clarity, using video-enhanced light mi- croscopy (Aliens al. 1981; Travis et al. 1983; Bowser & Rieder, 1985). Recent studies have demonstrated that the transport of internal organelles (Travis et al. 297