1. Generation of an erythrocyte vesicle transport system by Plasmodium falciparum malaria parasites
by Theodore F. Taraschi, Megan O'Donnell, Sandra Martinez, Timothy Schneider, Darin Trelka, Velia M. Fowler, Leann Tilley, and Yoshinori Moriyama
Blood
Volume 102(9):
November 1, 2003
©2003 by American Society of Hematology

2. Incubation of IRBCs with AlF reveals vesicle chains.
Incubation of IRBCs with AlF reveals vesicle chains. (A-B) Ultrastructural characterization of glutaraldehyde-fixed trophozoite stage IRBCs revealed vesicles with an electron-dense limiting membrane arranged in chains in the RBC cytosol (black arrowhead) or (C) closely apposed to the erythrocyte plasma membrane. A typical Maurer cleft is observed (A; white arrowhead). (D) Immunoelectron microscopy confirmed the long, slender unit membranes in the RBC cytosol are Maurer clefts. MC indicates Maurer cleft; P, parasite; PVM, parasitophorous vacuolar membrane; PPM, parasite plasma membrane, RBCM, red blood cell membrane; and V, vesicles. Scale bar = 70 nm.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Pf Sar1p is associated with coated vesicles and Maurer clefts.
Pf Sar1p is associated with coated vesicles and Maurer clefts. (A) Western blot analysis of uninfected and IRBCs incubated with AlF demonstrated that Pf Sar1p was an approximate 23-kDa Triton X-100-insoluble, SDS-soluble protein that was absent in uninfected RBCs. IFA showed Pf Sar1p localized to the parasite compartment and structures in the RBC cytosol. Immunoelectron microscopy revealed Pf Sar1p associated with (B) coated vesicles (V), black arrowhead, and (C) Maurer clefts (MC), white arrowhead, within the RBC cytosol. Scale bar = 70 nm.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Pf EMP1 is associated with coated vesicles and Maurer clefts.
Pf EMP1 is associated with coated vesicles and Maurer clefts. Trophozoite stage IRBCs were treated with AlF, processed for immunoelectron microscopy and probed with an α-Pf EMP1 antibody. Pf EMP1 associated with (A) the parasite compartment and coated vesicles in the RBC cytosol and (B) Maurer clefts. Scale bar = 70 nm.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

5. Pf NSF is associated with coated vesicles and Maurer clefts.
Pf NSF is associated with coated vesicles and Maurer clefts. (A) Western blot analysis of uninfected RBCs and IRBCs showed that Pf NSF was an approximate 89-kDa Triton X-100-insoluble, SDS-soluble protein that was absent in uninfected RBCs. Immunoelectron microscopy showed Pf NSF associated with (B) chains of coated vesicles in the RBC cytosol, black arrowhead, (C) coated vesicles closely apposed to the RBC membrane, black arrowhead, (D-E) the Maurer clefts, white arrowhead, (D) a large, double membrane structure in the RBC cytosol, and (E) transitional areas between a Maurer cleft and the RBC membrane. Scale bar = 70 nm.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Actin is associated with intraerythrocytic transport vesicles.
Actin is associated with intraerythrocytic transport vesicles. Trophozoite stage IRBCs were treated with AlF and processed for immunoelectron microscopy using an antiactin antibody. Actin associated with the RBC membrane and (A-B) coated vesicles in the RBC cytosol, (C) vesicle chains in the RBC cytosol closely apposed to the RBC membrane, and (D-E) vesicles (arrowhead) with a translucent limiting membrane and electron dense contents closely apposed to the RBC membrane. (F) Actin in close proximity to a coated vesicle budding from the PVM, black arrowhead, (G) concentrated in protruding areas of the PVM, black arrowhead, (H) associated with the Maurer clefts, which appeared to extend to actin-rich areas (black arrowheads) and a protruding, electron-dense area of the PVM (short arrow) that was similar in appearance (translucent limiting membrane and electron-dense contents) to the vesicles in panels D and E. (I) Maurer clefts in IRBCs permeabilized with streptolysin O maintained their parallel orientation and appeared to be tethered to the RBCs and parasite membranes via actin. Scale bar = 70 nm.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

7. RBC myosin association with the PVM appears to be G protein-mediated.
RBC myosin association with the PVM appears to be G protein-mediated. (A-B) In the absence of AlF, myosin was associated with the RBC membrane and PVM and diffusely distributed in the RBC cytosol. (C-D) AlF perturbs the myosin distribution to an almost exclusive association with the PVM and some structures in the RBC cytosol. (E-F) Immunogold EM revealed myosin to be associated with the PVM and Maurer clefts in AlF-treated IRBC. (G) Western blots showing that AlF causes a loss of myosin from the RBC membrane and stabilization at the PVM. In A-D, 1 mm equals 700 nm. Original magnification E-F, ×
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology

8. Proposed scheme for the transport of parasite proteins to the erythrocyte plasma membrane.
Proposed scheme for the transport of parasite proteins to the erythrocyte plasma membrane. (A) Many of the individual events depicted in Figures 1 to 5 are captured in a single (∼ 70 nm) section from an IRBC treated with AlF. Coated vesicles budding from the PVM form a vesicle chain that spans the RBC cytosol and associates with one end of a Maurer cleft. A vesicle in line with the (left-hand side) of a Maurer cleft is observed at the RBC membrane (white arrow). The Maurer cleft is anchored to the RBC plasma membrane via actin. Original magnification, × (B) Schematic representation of the transport of Pf EMP1 and Pf EMP3 (and other parasite proteins) to the RBC cytosol and plasma membrane. Steps 1 and 2 are hypothetical pathways. Pf Sec31, Pf Sar1p, and possibly Pf Sec23p (which is in the Pf genome but is uncharacterized) are exported to the RBC cytosol in which they form Pf COPII, facilitating vesicle formation at the PVM (steps 3-5). Vesicle budding and transport may be actin-myosin-mediated processes (step 5, 5a). Vesicles containing Pf EMP1 and Pf EMP3 uncoat (step 5) and are transported across the RBC cytosol to the Maurer clefts (step 6) or directly to the erythrocyte plasma membrane (step 6a) by an actin-myosin-mediated process. Pf NSF associates with the vesicles prior to their interaction with the clefts or the erythrocyte plasma membrane. Vesicles could be transported along actin-tethered Maurer clefts to the RBC membrane (step 7), or vesicles could bud from the ends of the Maurer cleft and diffuse to the RBC membrane. The vesicles associate with the RBC cytoskeleton (step 8) leading to knob formation.9 The appearance of extended chains of vesicles in the RBC cytosol and at the RBC membrane in IRBCs treated with AlF suggests an AlF-sensitive factor (eg, small GTPases such as Pfsar1p and/or Pf Rabs) may prevent vesicle uncoating (step 5) and block vesicle fusion at the clefts and/or erythrocyte plasma membrane, respectively, causing the vesicles to backup into chains.
Theodore F. Taraschi et al. Blood 2003;102:
©2003 by American Society of Hematology