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.Over 125 years after the discovery of its causative agent by Alphonse Laveran in 1880, malaria remains one of the most devastating infectious diseases in the world. With 300 -500 million cases and over a million deaths a year, this apicomplexan parasite represents a huge public health problem and a considerable economic burden (1). Malaria control efforts suffer from widespread resistance to anti-parasitic drugs and insecticides, underpinning the urgent need for novel intervention strategies.Transmission of malaria parasites starts with the ingestion of male and female gametocytes by vector mosquitoes during blood feeding on a parasite-infected host. Rapid gametogenesis and fertilization occur in the mosquito midgut, from which ookinetes develop that invade the midgut epithelium and transform into oocysts. After a two-week period of growth, mature oocysts release thousands of sporozoites into the mosquito hemolymph that invade the salivary glands of the insect and enter the vertebrate host during blood feeding to initiate new malaria infections.Invasive stages of malaria parasites, as well as related apicomplexan parasites, possess a unique cortical structure called the pellicle. This structure is made up of the plasma membrane, the inner membrane complex (IMC), 2 and subpellicular microtubules (2, 3). An additional structure of the pellicle, named the subpellicular ...

Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double null mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.

Graphical abstract

Graphical abstractThis paper describes the gametocyte-specific expression and subsequent targeting of two LCCL proteins to the crystalloids of Plasmodium berghei ookinetes using GFP-tagging.