Flagellar Membrane and Paraxial Rod Proteins of Leishmania: Characterization Employing Monoclonal Antibodies

Abstract

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.

Overexpression of S4D Mutant of Leishmania donovani ADF/Cofilin Impairs Flagellum Assembly by Affecting Actin Dynamics

“…As S4D protein-overexpressing cells were morphologically similar to the LdCof gene knockout mutants, in which PFR was absent (52), we analyzed the presence of the PFR in the S4D protein-overexpressing cells by both immunofluorescence microscopy and immunoblotting using MAb 2E10 (a kind gift of Diane McMahon Pratt, Yale University), which is known to selectively react with the two major Leishmania PFR proteins, viz., PFR1 and PFR2 (29). Whereas the flagella of the wild-type, LdCof-overexpressing, and S4A protein-overexpressing cells were intensely stained by MAb 2E10, the cells that overexpressed S4D protein remained completely unstained (Fig.…”

A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei

“…All coverslips were blocked with 3% BSA in PBS for 45 minutes, and then prepared for antibody labeling. YL1/2 (1:2000) (AbCam) L3B2 (1:25) (Kohl et al, 1999), anti-PAR (1:3000) (Ismach et al, 1989), anti-Centrin4 (1:500) (Shi et al, 2008) and anti-BiP (1:800) (Bangs et al, 1993) antibodies were used to label basal bodies, FAZ1 at FAZ filament, paraflagellar rod, bi-lobe (and basal bodies) and ER, respectively. The ratios in parentheses indicate the dilutions used for immunofluorescence assays.…”

Trafficking activity of myosin XXI is required in assembly ofLeishmaniaflagellum

“…We, therefore, examined the expression and location of PFR proteins in Myo21 +/-cells using mAb2E10 antibodies, which detect major PFR proteins, i.e. PFR1 and PFR2, in the flagellum of Leishmania promastigotes (Ismach et al, 1989). Unlike wild-type Leishmania promastigotes, no PFR staining was seen in Myo21 +/-cells (Fig.…”

“…Protein samples (cell lysates, 40 g/ lane) were resolved on SDS-PAGE and electroblotted on to a PVDF membrane. The blotted membranes were treated with blocking buffer (5% skimmed milk in PBS) and probed with the primary antibodies [actin, 1:10,000; Myo21, 1:1000; mAb10E2 (Ismach et al, 1989), 1:1000; GRP78 (Jensen et al, 2001), 1:100,000 and IFT172 (Absalon et al, 2008), 1:2000] diluted in blocking buffer. HRP-conjugated secondary antibodies (Santa Cruz) were used at 1:20,000 dilution.…”