9408635 Fulton Naegleria gruberi undergoes a developmental transformation from amoebae to flagellates over 100 minutes and may serve as a paradigm to study cell differentiation led us to calcium- binding proteins. Two calmodulins are synthesized during differentiation and become localized in flagellates, one in the flagella and the other in the flagellate cell body. Characterization of the single-copy intronless gene that encodes flagellar calmodulin (CaM-1) has shown that it cannot encode flagellate cell-body calmodulin (CaM-2). Initial attempts to isolate the CaM-2 gene led us to discover other differentiation-specific calcium-binding proteins, most notably centrin (a.k.a. caltractin). Our proposed research is focused on centrin and CaM-2. Centrin is a ubiquitous, diversely centriole-associated member of the calmodulin superfamily that has been implicated in Ca2+-mediated, ATPindependent contractility, a third contractile system. Although centrin is clearly a crucial eukaryotic protein, its functions, especially in relation to centrosome and basal body dynamics, are little understood. In Naegleria, centrin is developmentally regulated and becomes associated with the basal bodies when these organelles form de novo during differentiation. We plan to complete ongoing studies i) of the developmental regulation (e.g., phosphorylation) of centrin during Naegleria differentiation and basal-body assembly and ii) of remarkable structures formed by centrin-like proteins in certain other organisms, especially the spasmoneme of vorticellid ciliates. In order to better understand centrin, we plan iii) to seek and characterize proteins that associate with centrin using the cloned Naegleria centrin gene and the two-hybrid system in yeast and iv) to define by isolation, cloning and sequencing a centrin-related protein that is strongly implicated in contraction, the spasmin of Vorticella. In addition, we plan to define the second differentiation-specific calmodulin, CaM-2, by cloning its gene(s). These studies are of general interest in relation to two questions 1) What are the differences between the two differentiation-specific calmodulins of Naegleria, especially in relation to whether multiple calmodulins are a common feature among eukaryotes? 2) What is the structure and function of centrin, especially in Naegleria differentiation, what macromolecular structures does it form, and what is its relation to spasmin? Finally, a portion of our research effort continues to be directed toward obtaining DNA- mediated transformation of Naegleria, which would permit us to use the power of molecular genetics to dissect events during this otherwise ideal differentiation, such as the mechanism by which flagellar calmodulin (CaM-1) becomes localized in peninsular flagella. %%% This is a project which focuses on a group of protozoa on which very little work is currently being done, but which have evolved a fascinating interplay of cellular events. The protozoan, Naegleria gruberi, undergoes a dramatic shift from a crawling, amoeboid state to a swimming state by the de novo formation of flagella, the whip- like structures which motors many swimming cells. The work pursues this switch by concentrating on the new structural proteins which arise in the flagellated state, such as the contractile protein, centrin. In addition, by understanding the developmental control of genes which are newly transcribed, the switch to a new developmental pathway can be assessed. The switch may involve the expression of particular calcium binding proteins, or calmodulins. The project focuses on the characterization of the Naegleria centrin and the different forms of calmodulins. The association of centrins from Naegleria and another protozoan, Vorticella, with itself and other proteins is examined. The nature of this contractile protein is explored. The control of differential expression of calmodulins will be approached by testing the efficacy of obtai ning transgenic protists for these studies. The protists can then be transformed with different genetic variants of calmodulins and their effect on the development of flagella determined. By examining this unique organism, the diversity of microbes and their multiple ways of coping with their environment will be further illuminated. ***
