21The cyst wall of Entamoeba histolytica, the causative agent of the amoebiasis, is a 22 potential target for new drugs. The "Wattle and Daub" model of cyst wall formation of 23 Entamoeba invadens had already been reported. In this study, we demonstrate in more detail the 24 morphological stages of chitin wall formation in E. invadens using fluorescent chitin-binding 25 dyes and immunolocalization of the cyst wall proteins. Here, the expression and localization of 26 chitin synthase and the importance of actin cytoskeleton dynamics at cellular level, during 27 encystations have been demonstrated for the first time. Chitin deposition was found to be 28 initiated on the cell surface mostly from one distinct point, though multipoint initiation was also 29 observed sometimes. From these points, the wall grew outwards and gradually covered the entire 30 cyst surface with time. The initiation of chitin deposition was guided by the localization of chitin 31 synthase 1 on the plasma membrane. The gradual formation of the cyst wall follows the Wattle 32 and daub model. The chitin deposition occurred on the foundation of Jacob lectin at the cell 33 membrane, and the other cyst wall components, like chitinase, and Jessie were also found to be 34 present in the growing incomplete walls. In contrary to the Wattle and daub model, Jessie was 35 found to be expressed and localized in the growing wall at the early hours of encystations.
36During encystation, F-actin was reorganized into the cortical region within an hour encystation 37 initiation and remained intact until the completion of the chitin wall. Disruption of cortical actin 38 polymerization with 2, 3-Butanedione monoxime inhibited proper wall formation but produced 39 wall-less cysts or cysts with defective chitin wall. Malformations of cyst-walls were mainly due 40 to improper localization and activity of chitin synthases, which indicates the indispensability of 41 cortical actin cytoskeleton for the proper cyst wall formation.42 3 43 Author Summary 44 Entamoeba parasites reach new hosts using the resistant cyst form, so preventing its 45 formation can stop the spread of amoebiasis. The resistant nature of the cyst is due to the chitin 46 wall, and thus identifying the critical steps of wall formation could provide targets for designing 47 new drugs. Here we studied the morphological stages of the cyst wall formation by observing 48 how the chitin and other cell wall components were deposited on the cell surface using 49 fluorescent chitin-binding dyes and antibodies against cyst wall proteins. In most cases, the 50 chitin wall was found to start from one distinct point from which it spread all over the cell 51 surface, guided by chitin synthase. The composition of these incomplete walls was the same as a 52 mature cyst wall indicating that the wall may be a result of extracellular self-assembly of its 53 constituents from one starting point. We have also observed that F-actin polymerized in the 54 cortex of encysting cells and its disruption resulted in wall-less...