Characterization of Amoeba proteus myosin VI immunoanalog.
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.
Dominik
Magdalena
M
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Kłopocka
Wanda
W
Pomorski
Paweł
P
Kocik
Elzbieta
E
Redowicz
Maria Jolanta
MJ
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000656:Amoeba / Q000737:chemistry*; D000818:Animals; D000906:Antibodies; D034285:Dynamin II / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D020409:Molecular Motor Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment
D000199:Actins; D000906:Antibodies; D020409:Molecular Motor Proteins; C089785:myosin VI; D018995:Myosin Heavy Chains; D034285:Dynamin II