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IMC1b is a putative membrane skeleton protein involved in cell shape, mechanical strength, motility, and infectivity of malaria ookinetes.

Tremp AZ, Khater EI, Dessens JT - J. Biol. Chem. (2008)

Bottom Line: We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity.The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way.However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.

ABSTRACT
Membrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

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Differential expression of IMC1b. A, RT-PCR analysis of purified parasite samples enriched for asexual blood stages (asx), gametocytes (gct), ookinetes (ook), day 10 oocysts (ooc), and sporozoites (spz) for the presence of imc1b mRNA. Tubulin 1 was used as a reference gene. B, confocal microscope bright field and fluorescence images of different developmental stages. GFP (green) and DAPI (blue) images are combined.
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fig2: Differential expression of IMC1b. A, RT-PCR analysis of purified parasite samples enriched for asexual blood stages (asx), gametocytes (gct), ookinetes (ook), day 10 oocysts (ooc), and sporozoites (spz) for the presence of imc1b mRNA. Tubulin 1 was used as a reference gene. B, confocal microscope bright field and fluorescence images of different developmental stages. GFP (green) and DAPI (blue) images are combined.

Mentions: Life Stage Expression of IMC1b—We assessed transcription of the imc1b gene by RT-PCR of total RNA extracted from samples enriched for asexual blood stages, gametocytes, ookinetes, oocysts, and sporozoites, respectively. This assay revealed that transcription of the imc1b gene occurred predominantly in the enriched ookinete sample (Fig. 2A), pointing to an ookinete-specific expression of IMC1b. The expression of IMC1b protein was further studied using parasite line IMC1b/GFP. These parasites developed normally in mosquitoes and were readily transmitted by infected mosquito bites, demonstrating that the addition of the GFP tag to the carboxyl terminus of the IMC1b protein did not adversely affect parasite development. In support of the ookinete-specific IMC1b expression predicted from the imc1b transcription data (Fig. 2A), examination of IMC1b/GFP parasites by UV microscopy revealed strong GFP-based fluorescence in ookinetes, but not in asexual stages, oocysts, or sporozoites (Fig. 2B). GFP-based fluorescence in ookinetes was localized to the periphery of the cells (Fig. 2B), supporting a pellicular localization.


IMC1b is a putative membrane skeleton protein involved in cell shape, mechanical strength, motility, and infectivity of malaria ookinetes.

Tremp AZ, Khater EI, Dessens JT - J. Biol. Chem. (2008)

Differential expression of IMC1b. A, RT-PCR analysis of purified parasite samples enriched for asexual blood stages (asx), gametocytes (gct), ookinetes (ook), day 10 oocysts (ooc), and sporozoites (spz) for the presence of imc1b mRNA. Tubulin 1 was used as a reference gene. B, confocal microscope bright field and fluorescence images of different developmental stages. GFP (green) and DAPI (blue) images are combined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562075&req=5

fig2: Differential expression of IMC1b. A, RT-PCR analysis of purified parasite samples enriched for asexual blood stages (asx), gametocytes (gct), ookinetes (ook), day 10 oocysts (ooc), and sporozoites (spz) for the presence of imc1b mRNA. Tubulin 1 was used as a reference gene. B, confocal microscope bright field and fluorescence images of different developmental stages. GFP (green) and DAPI (blue) images are combined.
Mentions: Life Stage Expression of IMC1b—We assessed transcription of the imc1b gene by RT-PCR of total RNA extracted from samples enriched for asexual blood stages, gametocytes, ookinetes, oocysts, and sporozoites, respectively. This assay revealed that transcription of the imc1b gene occurred predominantly in the enriched ookinete sample (Fig. 2A), pointing to an ookinete-specific expression of IMC1b. The expression of IMC1b protein was further studied using parasite line IMC1b/GFP. These parasites developed normally in mosquitoes and were readily transmitted by infected mosquito bites, demonstrating that the addition of the GFP tag to the carboxyl terminus of the IMC1b protein did not adversely affect parasite development. In support of the ookinete-specific IMC1b expression predicted from the imc1b transcription data (Fig. 2A), examination of IMC1b/GFP parasites by UV microscopy revealed strong GFP-based fluorescence in ookinetes, but not in asexual stages, oocysts, or sporozoites (Fig. 2B). GFP-based fluorescence in ookinetes was localized to the periphery of the cells (Fig. 2B), supporting a pellicular localization.

Bottom Line: We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity.The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way.However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.

ABSTRACT
Membrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

Show MeSH
Related in: MedlinePlus