Patterning of Cellular Differentiation by Lateral Inhibition in a Filamentous Cyanobacterium

从一组等同细胞发展出分化细胞类型的模式是一种基本现象。鱼腥藻菌株PCC 7120是一种丝状蓝藻，可以被诱导从一系列未分化的营养细胞分化出一种固氮异形胞模式。 异形胞平均每隔10个细胞出现一次，是终末分化的，在形态、代谢和遗传上与营养细胞不同。 它们允许在空间上分离光合作用和固氮这两个不相容的过程。 分化模式似乎取决于几种蛋白质之间的相互作用。 第一个是HetR，它是调节电路的一部分，具有生物开关的特性，将分级输入信号转换为二进制输出：当开关“关闭”时，细胞保持未分化，但当开关“打开”时，分化过程开始并最终变得不可逆和自我维持。 HetR起促进分化的作用，并且是诱导分化所必需的和充分的。 PatS是一种阻止分化的蛋白质，显然是通过与HetR的相互作用，并负责决定细丝上异型胞的从头图案化。HetN产生一个信号，参与模式一旦形成后的稳定和维持。 前提是细胞的相对位置由从分化源细胞延伸的PatS和/或HetN的浓度梯度传递。 这种“侧抑制”现象与许多发育系统中的细胞分化和模式化有关;但这在相对较少的系统中得到了实验证明。 有大量的初步证据表明，HetN和PatS依赖的信号从一个细胞到另一个细胞在鱼腥藻的细丝中移动，以产生分化的异形胞的周期性模式，并在中间异形胞生长和分裂时维持它。 HetN的五肽RGSGR是其作为模式蛋白的功能所必需的。该肽也存在于PatS中，并且认为这些肽积极参与细胞分化的抑制。然而，HetN的功能形式是未知的，因为这两种抑制剂的细丝内的细胞间运动的途径。因此，该项目的两个具体目标是：1。表征HetN的功能形式，其从细胞扩散到细胞以维持异形胞模式，以及2.确定HetN和PatS依赖的抑制信号的细胞间转移的手段。这个项目是狭隘地集中在蓝藻异形胞分化的遗传学，但更广泛的生物学影响将是在许多生物体中控制细胞分化的分子机制的类型。 此外，该项目还将通过资助研究生和本科生的研究培训、加强本科生遗传学实验室课程的各部分、向受资助的研究生提供教学机会以及允许他们参加国家会议，促进教学、培训和学习。 指导本科生获得研究学分，并作为少数民族进入研究职业（MARC）计划的一部分，将有助于扩大太平洋岛民后裔学生在生物科学中的参与。 此外，蓝藻开始被用于工业目的。 人们希望现有的产生额外异形胞的转基因菌株能够增加蓝藻固氮的副产品生物氢的产量。 不幸的是，与野生型菌株相比，这些菌株不固定额外的氮，也不产生额外的氢，因为额外的异形胞由于缺乏营养细胞的边界而聚集在一起。 为了设计一种单个异形胞之间间距减小的菌株，需要了解HetN和PatS如何确定异形胞的周期性模式，这是本提案中描述的工作的目的。

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.