Patterning of Cellular Differentiation by Lateral Inhibition in a Filamentous Cyanobacterium

University Of Hawaii, Honolulu

Investigators

Abstract

The development of a pattern of differentiated cell types from a group of equivalent cells is a fundamental phenomenon. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can be induced to differentiate a pattern of nitrogen-fixing heterocysts from a chain of undifferentiated vegetative cells. Heterocysts occur, on average, at 10 cell intervals, are terminally differentiated, and differ from vegetative cells morphologically, metabolically, and genetically. They allow the spatial separation of the two incompatible processes of photosynthesis and nitrogen fixation. Patterning of differentiation appears to be dependent on the interactions between several proteins. The first, HetR, is part of a regulatory circuit that shares the properties of biological switches, which turn graded input signals into a binary output: when the switch is "off",the cell remains undifferentiated, but when the switch is "turned on", the differentiation process begins and eventually becomes irreversible and self-sustaining. HetR acts to promote differentiation and is both necessary and sufficient to induce differentiation. PatS is a protein that prevents differentiation, apparently through interaction with HetR, and is responsible for determining the de novo patterning of heterocysts on a filament. HetN produces a signal involved in stabilization and maintenance of the pattern once it has formed. The premise is that the relative positions of cells is conveyed by concentration gradients of PatS and/or HetN extending from differentiating source cells. This phenomenon of "lateral inhibition" is implicated in governing the cellular differenitation and patterning in many developmental systems; but this has been experimentally demonstrated in relatively few systems. There is abundant preliminary evidence that HetN- and PatS-dependent signals move from cell to cell in filaments of Anabaena to create the periodic pattern of differentiated heterocysts and maintain it as the intervening heterocysts grow and divide. Only a pentapeptide, RGSGR, of HetN is required for its function as a patterning protein. This peptide is present also in PatS and is thought that these peptides are actively involved in the the suppresion of cellular differentiation. However, the functional form of HetN is unknown as is the route of intercellular movement within filaments of both inhibitors. Thus, the two specific aims of this project are to: 1. characterize the functional form of HetN that diffuses from cell to cell to maintain heterocyst patterning, and 2. determine the means of intercellular transfer of both HetN- and PatS-dependent inhibitory signals. This project is narrowly focused on the genetics of heterocyst differentiation in cyanobacteria, but the broader biological impact will be on the types of molecular mechanisms that control specification of cells for differentiation in many organisms. In addition, the project will promote teaching, training and learning by funding the research training of graduate students and undergraduate students, by enhancing sections of an undergraduate genetics laboratory course, by providing funded graduate students with teaching opportunities and by allowing their participation at national meetings. Mentoring of undergraduate students for research credit and as part of the Minority access to Research Careers (MARC) program will help to broaden participation of students of Pacific Islander descent in the biological sciences. In addition, cyanobacteria are beginning to be exploited for industrial purposes. It was hoped that existing genetically modified strains that produce extra heterocysts would have increased biohydrogen production, a byproduct of nitrogen fixation, by cyanobacteria. Unfortunately, these strains do not fix extra nitrogen nor produce extra hydrogen compared to the wild type strain because the extra heterocysts are clustered together for lack of bordered by vegetative cells. To engineer a strain with reduced spacing between individual heterocysts, one needs to understand how HetN and PatS determine the periodic pattern of heterocysts, which is the purpose of the work described in this proposal.