Evolution of a Fundamental Morphogenetic Process in the Volvocine Algae

我们的目标是确定一个必要的形态发生过程是如何出现的，需要广泛的细胞间协调，多细胞团藻从单细胞祖先进化而来，类似于它最近的单细胞亲戚衣藻。为了能够游泳，在胚胎发生结束时，团藻必须在一个类似原肠胚形成的过程中向右翻转，这个过程被称为“反转”，这涉及到协调的细胞骨架重排和细胞形状和运动的变化。带有各种反转缺陷的转座子标记突变体已经产生。其中第一个被分析的是Inv61，在其中，由于转座子插入到invA中的结果，反转在中途被阻止，invA是一种编码一种新的运动蛋白的基因，该蛋白位于胚胎中靠近细胞质桥的地方，在反转中起关键作用。我们克隆了C. reinhardtii invA （iarA）的同源基因，发现其编码的运动蛋白（iarA）在其运动和尾部结构域的aa序列中与invA相同90%。因为invA编码微管马达，而invA突变体类似于暴露于肌动球蛋白抑制剂的野生型胚胎，我们将探索invA在Volvox中作为肌动蛋白和微管蛋白细胞骨架之间的联系的可能性。同时，我们将研究其同源物IarA在整个衣藻细胞周期中的表达和定位，并将研究使用显性阴性结构体、抗体抑制或RNA干扰阻断IarA功能的影响。我们将使用核转化来确定iarA是否可以拯救invA突变体。如果没有，我们将测试嵌合的IarA - InvA结构来定义ina的区域，这些区域需要赋予IarA反转功能。这些结构差异的功能意义将随后通过适当的运动蛋白结合和/或运动分析进行测试。如果iarA确实拯救了invA，我们将比较衣藻中与iarA相互作用的蛋白质和与它相互作用的Volvox中的蛋白质。

0131565KirkOur goal is to determine how an essential morphogenetic process requiring extensive intercellular coordination arose as multicellular Volvox evolved from a unicellular ancestor resembling its closest unicellular relative, Chlamydomonas. In order to be able to swim, at the end of embryogenesis Volvox must turn right-side-out in a gastrulation-like process called "inversion," which involves coordinated cytoskeletal rearrangements and changes in cell shape and motility. Transposon tagged mutants with a variety of inversion (inv) defects have been generated. The first of these to be analyzed was Inv61, in which inversion is arrested at the halfway point as a result of a transposon insertion in invA, a gene encoding a novel kinesin that is localized in the embryo near the cytoplasmic bridges that play a key role in inversion. We have cloned the C. reinhardtii orthologue of invA (iarA) and found that it encodes a kinesin (IarA) that is 90 % identical in aa sequence to InvA in both its motor and tail domains. Because invA encodes a microtubule motor, whereas invA mutants resemble wild-type embryos exposed to actomyosin inhibitors, we will explore the possibility that InvA acts as a link between the actin- and tubulin-based cytoskeletons in Volvox. Meanwhile, we will study expression and localization of its orthologue, IarA, throughout the Chlamydomonas cell cycle, and will study the effects of blocking IarA function, using a dominant negative construct, antibody inhibition, or RNA interference. We will use nuclear transformation to determine whether iarA can rescue the invA mutant. If it does not, we will test chimeric iarA-invA constructs to define regions of InvA that are required to confer an inversion function on IarA. The functional significance of such structural differences will then be tested with appropriate kinesin binding and/or motility assays. If iarA does rescue invA, we will compare the proteins with which IarA interacts in Chlamydomonas versus those with which it interacts in Volvox.