title: Overexpression of S4D mutant of Leishmania donovani ADF/cofilin impairs flagellum assembly by affecting actin dynamics.
abstract: Leishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmania actin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum.
authors.affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Kumar
authors.forename: Rashmi
authors.initials: R
authors.lastname: Srivastava
authors.forename: Kalyan
authors.initials: K
authors.lastname: Mitra
authors.forename: Amogh A
authors.initials: AA
authors.lastname: Sahasrabuddhe
authors.forename: Chhitar M
authors.initials: CM
authors.lastname: Gupta
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D051338:Cofilin 1 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000378:metabolism* / Q000648:ultrastructure; D042541:Intracellular Space / Q000378:metabolism; D007893:Leishmania donovani / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D010641:Phenotype; D058105:Polymerization; D011485:Protein Binding; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D051338:Cofilin 1; D009711:Nucleotides; D015800:Protozoan Proteins; D001224:Aspartic Acid; D012694:Serine