Characterization of Amoeba proteus myosin VI immunoanalog

Abstract

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

Dynamin as a mover and pincher during cell migration and invasion

“…Injection of anti-dynamin antibodies into this organism results in a loss of directional migration (Dominik et al, 2005) as well as a reduction in the rate of uroidal translocation. Amoeba Dyn2 appears to precipitate with actin pellets in an ATP-independent manner, which emphasizes its role in actin dynamics.…”

Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy

“…12 In this paper, we use SDPM to perform Doppler flow imaging of cytoplasmic streaming in an Amoeba proteus, which is very mobile unicellular protozoan that has been widely used for decades to study the mechanisms of cytoplasm physiology and cell motility. [13][14][15][16][17][18] Cytoplasmic streaming is an important cellular process that plays a role in events ranging from metastasis in cancer to cellular locomotion to cell division. We derive an expression for the shot-noise-limited flow velocity resolution of SDPM, and compare the performance of our system to this theoretical limit.…”

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

“…The results presented here show that a novel A. proteus myosin motor, which we detected earlier (Dominik et al 2005), plays an important role during phagocytosis and pinocytosis, the indispensable processes of intracellular vesicular transport and reorganization of the actin cytoskeleton.…”

“…1), the same one detected earlier by several antibodies developed against various tail regions of human and chicken myosin VI (Dominik et al 2005). To characterize the involvement of the myosin VI immunoanalog in pinocytosis and phagocytosis, we assessed not only localization of this protein in pinocytotic and phagocytotic amoebae, but also the effect of blocking endogenous protein on these processes.…”

“…It is known that incubation of amoebae with 125 mmol/L NaCl or KCl causes contraction of actin cytoskeleton beneath the plasma membrane, leading to rosette-shaped cells protruding pinocytotic pseudopodia with pinocytotic channels (Stockem et al 1983;Kłopocka and Grębecka 1985;Dominik et al 2005). After a 15 min incubation period with 125 mmol/L NaCl, amoebae were subjected to an immunostaining procedure.…”