Research: J.L. Travis

STRUCTURAL AND FUNCTIONAL IMPLICATIONS OF AN UNUSUAL FORAMINIFERAL β-TUBULIN

A. Habura, L.Wegener, J.L. Travis, and S. S. Bowser

Wadsworth Center, New York State Department of Health, Albany, New York
Department of Biological Sciences, State University of New York at Albany

We have obtained sequence data for β-tubulin genes from eight species of Foraminifera (forams) and α-tubulin sequences from four species, sampling major taxonomic groups from a wide range of environments. Analysis of the β-tubulin sequences demonstrates that foram β-tubulins possess the highest degree of divergence of any tubulin gene sequenced to date and represent a novel form of the protein. In contrast, foram α-tubulin genes resemble the conventional α-tubulins seen in other organisms. Partition homogeneity analysis shows that the foraminiferal β-tubulin gene has followed an evolutionary path that is distinct from that of all other organisms. Our findings indicate that positive selective pressure occurred on the β-tubulin subunit in ancestral forams prior to their diversification. The specific substitutions observed have implications for microtubule (MT) assembly dynamics. The regions most strongly affected are implicated in lateral contacts between protofilaments and in taxol binding. We predict that these changes strengthen lateral contacts between adjacent dimers in a manner similar to that induced by taxol binding, thus allowing the formation of the tubulin “helical filaments” observed in forams by electron microscopy. Our results also indicate that substantial changes to these portions of the β-tubulin molecule can be made without sacrificing essential MT functions.

REACTIVATION OF CELL SURFACE TRANSPORT IN Reticulomyxa.
D.D. Orokos, S.S. Bowser, and J.L. Travis.
Department of Biological Sciences, University at Albany, SUNY, Albany, NY 12222 and
Wadsworth Center, P.O. Box 509, Albany, NY 12201

Granuloreticulosean protists transport particles (e.g. bacteria, algae, and sand grains) along the outer surfaces of their pseudopodia. This cell surface transport plays a vital role in feeding, reproduction, shell construction and locomotion and can be visualized by the movements of extracellularly-adherent polystyrene microspheres (i.e. latex beads). Our videomicroscopic analyses of transport associated with the pseudopodia of Reticulomyxa filosa revealed two distinct types of both intracellular and cell surface transport: (i) saltatory, bidirectional transport of individual or clustered organelles and / or surface-attached particles, and (ii) continuous, unidirectional bulk or "resolute" motion of aggregated organelles and / or surface-bound particles. Organelles and surface-attached polystyrene microspheres remained firmly attached to the microtubule cytoskeletons of detergent-extracted pseudopodia. Both saltatory and resolute organelle and surface transport reactivated upon the addition of 0.01 - 1.0 mM ATP. At 1 mM ATP, the velocities of reactivated saltatory transport were indistinguishable from those observed in vivo. The reactivated transport was microtubule-dependent and was not inhibited by incubation with Ca2+-gelsolin under conditions that abolish rhodamine-phalloidin detection of actin filaments. These findings provide further support that both intracellular organelle and membrane surface transport are mediated by a commom mechanism, and establish Reticulomyxa as a unique model system to further study the mechanochemistry of cell-surface transport in vitro.

CELL SURFACE AND ORGANELLE MOTILITY SHARE THE SAME ENZYMATIC PROPERTIES IN Reticulomyxa.
D.D. Orokos and J.L. Travis.
Department of Biological Sciences, University at Albany, Albany, NY 12222.

Reticulomyxa transports particulates, like bacterial and algal prey items, bidirectionally along the outside of its pseudopodia. This cell surface transport and intracellular organelle transport can be reactivated in detergent permeabilized cell models [Orokos et al, 1997. Cell Motil. Cytoskel. 37: 139 - 148]. We have used this unique system to compare cell surface and organelle mechanochemistry in situ in the same reactivated pseudopodia. The ATPase activities of both types of transport were indistinguishable; each displayed identical nucleoside triphosphate specificity, transport ATPase kinetics, and inhibitor sensitivity. Organelle and cell surface transport reactivation required "hydrolyzable" adenosine nucleoside triphosphates; neither reactivated with GTP, CTP, UTP, ITP, AMP-PNP, AMP-PCP or ATP-gamma-S. However, other ATP analogues, such as 2'-deoxy ATP and 3'-deoxy ATP and 2',3'-dideoxy ATP supported the reactivation of organelle and cell surface transport at similar, but markedly reduced, velocities. Both transport processes were inhibited similarly by known inhibitors of dynein ATPases such as EHNA or Na-orthovanadate. NEM and UV irradiation in the presence of Na-orthovandate and ATP permanently disabled both transport processes. Organelle and surface transport followed identical Michaelis-Menten kinetics with a calculated Km of 118 mM ATP and a maximum translocation velocity (Vmax) of 8.33 mm/sec. These findings strongly suggest that cell suface transport shares the same cytoplasmic dynein motor [Schliwa et al, 1991. J. Cell Biol. 112: 1199 - 1203] that drives organelle transport.

ORGANELLES ARE TRANSPORTED ON SLIDING MICROTUBULES IN Reticulomyxa
D.D. Orokos, R.W. Cole and J.L. Travis.
Department of Biological Sciences, University at Albany, Albany, NY 12222 and
Wadsworth Center, B.O. Box 509, Albany, NY 12201

Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15 mm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 minutes and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

AUTONOMOUS REORGANIZATION OF FORAMINIFERAN RETICULOPODIA
J.L. Travis, E.A. Welnhofer and D.D. Orokos.
Department of Biological Sciences, University at Albany, Albany, NY 12222 and
Department of Biology, Canisius College, Buffalo, NY 14208

Reticulopodia, the diagnostic cytoplasmic appendage of the foraminifera, are complex networks of branched and anastomosed pseudopodia. The instantaneous patterns of these networks are highly variable and are remodeled almost continuously. Real-time light microscopic observations demonstrate that these morphogenetic processes occur autonomously throughout the entire networks, even at their farthest reaches which may be located at great distances from the cell body. Portions of reticulopodia severed from the cell body undergo autonomous and stereotyped morphological rearrangements to form satellites, consist of a relatively large cytoplasmic mass located centrally within a radiating pseudopodial network. Pseudopodial movements and structural rearrangements are active energy-requiring processes that depend on intracellular factors such as the formation and reorganization of microtubule cytoskeletal elements. Here we show that that reticulopodia consistently assume a spiral shape when placed on a cationic substrate, indicating that the pseudopodial pattern is also influenced by environmental factors such as interactions with the substratum. Our studies suggest that reticulopodia are self-organizing, and that their instantaneous reticular form is an emergent property continuously recalculated from real-time processing of myriad physiological and environmental imputs. Reticulopodial form at any point or region appears to be the summed behavioral response to these parameters. Our studies indicate that reticulopodial morphology and behavior can be modified by environmental stimuli, like substratum adhesivity, and suggest that processes, such as shell patterning, that depend on pseudopodia could also be modified by environmental factors.

EVIDENCE FOR A DIRECT CONVERSION BETWEEN TWO TUBULIN POLYMERS - MICROTUBULES AND HELICAL FILAMENTS - IN THE FORAMINIFERAN, Allogromia laticollaris
E.A. Welnhofer and J.L. Travis.
Department of Biological Sciences, University at Albany, Albany, NY 12222.

In Allogromia, tubulin lattices transform between the microtubule and helical filament states. Helical filaments are composed of ~10 nm thick tubulin filaments wound into ~30 nm dia. coils. The pathway of the transitions between these two lattice states was examined in vitro in detergent-lysed reticulopodia. Microtubules represented the majority of the assembled tubulin polymers in the detergent extracted pseudopodia, however these transformed into helical filaments upon exposure to 10 mM Ca2+ or 50 mM Mg2+. The conversion of microtubules into helical filaments involved fragmentation of the tubulin lattice and reduction in total polymer length. Divalent cations were required for the maintenance of the helical filament state; their removal resulted in the loss of helical filaments and the formation of microtubules. The observation that the lattice state transitions occurred in vitro in the lysed cells suggests that they occur independently of soluble tubulin concentration, and supports a direct transition model in which the tubulin lattice interconverts between the helical filament and microtubule states. We propose a structural model for the direct pathway whereby disruption of longitudinal bonds between tandem tubulin dimers in protofilaments causes the microtubule lattice to unwind into helical filaments -- continuous ribbons of laterally connected tubulin dimers- from the microtubule end. Helical filaments may wind into microtubule as the longitudinal intersubunit bonds reform.