Regulation of Drosophila larval fat body development and function by tissue-specific SPARC variants

基底膜（BM）在所有多细胞生物的发育、生长、再生能力和组织稳态中起着不可或缺的作用。BM的核心成分包括网络形成胶原蛋白IV（ColIV）。然而，控制BM组装的分子过程知之甚少。BM组装和重塑需要非核心组件，通常以组织特异性方式进行。在我的实验室使用果蝇的工作已经建立了一个必不可少的分子伴侣ColIV，使非聚合的ColIV从其生产的中心网站在幼虫的脂肪体扩散到远端网站，如翼成虫盘。我们已经表明，损失的脂肪体和2龄幼虫致死率内的ColIV纤维样积累的结果。它由酸性钙结合N端结构域、中央卵泡抑素样结构域和C端结构域组成，C端结构域含有两个高度保守的胶原结合表位和两个高亲和力钙结合EF-手。高度保守的胶原蛋白结合表位和EF-手的可识别性，使Col与ColIV的关联。我们确定，在ColIV结合能力的损失是负责幼虫致死。此外，脂肪体中表达缺失足以致死。重要的是，翼盘产生的脂肪能够迁移到脂肪体，但不与任何组织中的BM相关联，这意味着来自这些组织的脂肪的不同作用。我们确定，ESTA第一共定位与ColIV内的反式高尔基体的血细胞和脂肪体脂肪细胞表明在防止细胞内ColIV聚合的作用。总的来说，我们的数据提供了第一个体内证据表明，不同的组织表达不同的变体，对BM动力学，脂肪体功能和幼虫发育有不同的影响。我们假设，翼成虫盘衍生的脂肪体，它具有类似的分子量的脂肪体分泌的脂肪，代表一种糖型不能结合ColIV和脂肪体产生的不同的功能。在这个建议中，提出了一系列的策略，继续利用果蝇的遗传易处理性和分子资源，以获得进一步的机制洞察到不同的形态调节的贡献，果蝇幼虫发育过程中。我们迄今的工作提供了强有力的证据，证明ESTs作为Col（IV）的伴侣分子，能够实现适当的组织分布。然而，关键的机制问题仍然需要解决，以完全接受这种独特的功能分泌的细胞外蛋白。本提案中概述的工作将提供这些信息，并将检验我们的假设，即翅成虫盘衍生的蛋白质代表一种不能结合Col（IV）的糖型，并发挥与脂肪体衍生的蛋白质不同的功能。

Basement membranes (BMs) play an indispensable role in development, growth, regenerative capacity, and tissue homeostasis of all multicellular organisms. A core component of BMs includes network-forming Collagen IV (ColIV). However, the molecular processes controlling the assembly of BMs are poorly understood. Non-core components are required for BM assembly and remodelling, often in a tissue-specific manner. Work in my laboratory using Drosophila has established SPARC as an essential molecular chaperone for ColIV, enabling diffusion of non-polymerized ColIV from its central site of production in the larval fat body to distal sites, such as the wing imaginal discs. We have shown that loss of SPARC results in fibrotic-like accumulation of ColIV within the fat body and 2nd instar larval lethality. SPARC consists of an acidic calcium-binding N-terminal domain, a central follistatin-like domain, and a C-terminal domain containing two highly conserved collagen-binding epitopes and two high-affinity calcium-binding EF-hands. The highly conserved collagen-binding epitopes and EF-hands of SPARC enable association of SPARC with ColIV. We determined that a loss in the ColIV-binding capacity of SPARC is responsible for the larval lethality. Moreover, loss of SPARC expression in the fat body is sufficient for lethality. Importantly, SPARC generated by the wing imaginal disc is able to migrate to the fat body, but does not associate with BMs in either tissue, implying distinct roles for SPARC derived from these tissues. We determined that SPARC first colocalizes with ColIV within the trans-Golgi of hemocytes and fat body adipocytes indicating a role in preventing intracellular ColIV polymerization. Collectively, our data provide the first in vivo evidence that different variants of SPARC are expressed by tissues with distinct impact on BM dynamics, fat body function and larval development. We hypothesize that wing imaginal disc-derived SPARC, which has a similar molecular weight to that of SPARC secreted by the fat body, represents a glycoform incapable of binding ColIV and with distinct functions from that produced by the fat body. In this proposal, a series of strategies are proposed that continue to take advantage of the genetic tractability and molecular resources of Drosophila to gain further mechanistic insight into the diverse morphoregulatory contributions of SPARC during larval development. Our work to date provides strong evidence that SPARC functions as a chaperone for Col(IV) enabling appropriate tissue distribution. However, key mechanistic questions remain that need to be addressed to fully accept this unique function of a secreted extracellular protein. The work outlined in this proposal will provide this information and will test our hypothesis that wing imaginal disc-derived SPARC represents a glycoform incapable of binding Col(IV) and exerts distinct functions from that produced by the fat body-derived SPARC.