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

基底膜（BMs）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。脑转移瘤的核心成分包括形成网络的胶原IV （ColIV）。然而，控制脑转移瘤组装的分子过程尚不清楚。BM装配和重塑需要非核心组件，通常以组织特定的方式。在我的实验室中，通过对果蝇的研究发现，SPARC是大肠杆菌必不可少的分子伴侣，可以使未聚合的大肠杆菌从其在幼虫脂肪体中的中心生产部位扩散到远端部位，如翅膀的成像盘。我们已经证明SPARC的缺失会导致ColIV在脂肪体内的纤维样积累和2龄幼虫的致命性。SPARC由一个酸性钙结合n端结构域、一个中央卵泡抑素样结构域和一个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 -手的c端结构域组成。SPARC的高度保守的胶原结合表位和ef -hand使SPARC与ColIV相关。我们确定SPARC的大肠杆菌结合能力的丧失是导致幼虫死亡的原因。此外，脂肪体中SPARC表达的缺失足以致死性。重要的是，由翅膀想象盘产生的SPARC能够迁移到脂肪体，但不与任何组织中的脑转移相关，这意味着来自这些组织的SPARC具有不同的作用。我们确定SPARC首先在血细胞和脂肪体脂肪细胞的反式高尔基体中与ColIV共定位，表明其在阻止细胞内ColIV聚合中的作用。总的来说，我们的数据提供了第一个体内证据，表明SPARC的不同变体在组织中表达，对BM动力学、脂肪体功能和幼虫发育有不同的影响。我们假设翼想象盘衍生的SPARC与脂肪体分泌的SPARC具有相似的分子量，是一种不能结合ColIV的糖型，与脂肪体产生的SPARC具有不同的功能。本文提出了一系列策略，继续利用果蝇的遗传易变性和分子资源，进一步深入了解SPARC在幼虫发育过程中的多种形态调控作用。迄今为止，我们的工作提供了强有力的证据，证明SPARC作为Col（IV）的伴侣，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，以充分接受分泌的细胞外蛋白的这种独特功能。本提案中概述的工作将提供这一信息，并将验证我们的假设，即翼想象盘衍生的SPARC代表一种不能结合Col（IV）的糖型，并且与脂肪体衍生的SPARC产生的糖型具有不同的功能。

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.