Microtubule associated motor proteins of Plasmodium falciparum merozoites

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Abstract

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to γ tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.

Introduction

Plasmodium falciparum and other apicomplexans have been shown to invade cells using an acto-myosin-based motor [1], [2], [3]. However, depolymerisation of microtubules (mt) in P. falciparum merozoites, has also been shown to inhibit invasion [4], [5]. The P. falciparum merozoite assemblage of sub-pellicular mt (f-MAST) runs underneath the cisternal membranes, from the apical rings, terminating near the nucleus in the basal region of the cell. This narrow band of two or three parallel mt forms a single strut [5], [6], [7]. During merogony, the period of merozoite formation, the f-MASTs radiate from centrally positioned nuclei, which surround the residual body. Depolymerisation of these structures, using dinitroaniline compounds, reduced invasion rates in assays initiated with post-mitotic schizonts, whereas stabilising mt with taxol had no effect on invasion rates [5].

Why the integrity of these structures is important to merozoite function remains open to question. There are several potential roles, corollaries of known mt function in other cells. Microtubule based motor proteins, of which the most extensively studied are the kinesin family and cytoplasmic dyneins (c dynein), are known to be involved in various types of intracellular transport [8], [9], [10], [11]. Classically, most kinesins shuttle cargo towards mt plus ends, i.e. where rapid addition of tubulin sub-units occurs, and c dynein carries cell traffic in the opposite direction, i.e. towards mt minus-ends, where the nucleation of mt begins. γ-tubulin forms sites for mt nucleation and minus end anchorage [12], [13]. Therefore, we have used γ-tubulin immunofluorescence to determine the polarity of the f-MAST.

To elucidate the roles of f-MASTs in merogony and the invasion process, we used Western blotting, fluorescent imaging (FI) and invasion assays to investigate kinesin and c dynein in the P. falciparum asexual cycle. We discuss the potential roles for these motor proteins in the biology of P. falciparum merozoites, and their relevance to RBC invasion.

Section snippets

Parasite culture and western blots

P. falciparum (C10 strain) was maintained in continuous culture using standard medium, atmosphere and techniques (see [14]). Cultures were synchronised to the appropriate life-cycle stages using sorbitol lysis [15] or Percoll® density cushions [16]. CF11 cellulose (Whatman) columns were used to remove white blood cells, so that host motor-proteins and tubulins were excluded. Saponin (0.05%) lysis released rings from erythrocytes in the presence of a cocktail of protease inhibitors (see [14]).

Kinesin western blot

Using an antibody to the KHC of conventional kinesin, immunoblotting of merozoite proteins showed a clear band at 135 kDa (Fig. 1). The same antibody showed strong bands of the same size on blots of proteins from four stages of the asexual lifecycle; rings, trophozoites, schizonts and purified merozoites (Fig. 2). Two heavy bands were also clearly detected at about 210 kDa in the ring-stages. These may represent ubiquitinated or precursor molecules, or possibly cross-reactivity of the antibody

Discussion

We have demonstrated, for the first time, the presence of the microtubule motor proteins, kinesin and dynein in P. falciparum merozoites. The kinesins form a large family of proteins, with different tasks within the cell [26]. The K 1005 antibody may have affinity for more than one type of kinesin, which might explain the strong signals and variety of FI patterns seen in this study. Cytoplasmic dynein, of which there at least three types [27], has a multi-chain structure and also complexes with

Acknowledgements

The authors gratefully acknowledge the support of this work by the Wellcome Trust: grant numbers 048244 & 059566, The Special Trustees of Guy's and of St Thomas's Hospital and equipment grants from The Royal Society. They are grateful to Ann Taylor and Joel Rosenbaum for suggestions and stimulating discussion. We wish to thank the scientists and funding agencies comprising the international Malaria Genome Project for making sequence data from the genome of P. falciparum (3D7) public prior to

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