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

基底膜在所有多细胞生物体的发育、生长、再生能力和组织动态平衡中起着不可或缺的作用。BMS的核心成分包括形成网络的IV型胶原(ColIV)。然而，控制BMS组装的分子过程却鲜为人知。非核心组件是BM组装和重建所必需的，通常是以特定于组织的方式进行的。在我的实验室中使用果蝇的工作已经建立了SPARC作为ColIV的基本分子伴侣，使非聚合的ColIV能够从其在幼虫脂肪体中的中心产生部位扩散到远端部位，如翅膀成像盘。我们已经证明，SPARC的缺失会导致脂肪体内胶原IV的纤维性堆积，并导致2龄幼虫的死亡。SPARC由一个酸性钙结合N-末端结构域、一个中央卵泡抑素样结构域和一个C-末端结构域组成，C-末端结构域包含两个高度保守的胶原结合表位和两个高亲和力的钙结合EF-HAND。SPARC的高度保守的胶原结合表位和EF-HAND使SPARC能够与ColIV结合。我们确定SPARC与ColIV结合能力的丧失是导致幼虫死亡的原因。此外，脂肪体内SPARC表达的缺失足以致死。重要的是，翅膀形象盘产生的SPARC能够迁移到脂肪体内，但与这两个组织中的BMS无关，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跨高尔基体内与ColIV共定位，这表明了SPARC在防止细胞内ColIV聚合中的作用。总之，我们的数据提供了第一个活体证据，表明不同的SPARC变体在组织中表达，对骨髓动力学、脂肪体功能和幼虫发育有不同的影响。我们推测，翼想象盘源性SPARC与脂肪体分泌的SPARC分子量相似，代表一种不能结合ColIV的糖形式，具有与脂肪体产生的糖形式不同的功能。在这项建议中，提出了一系列策略，以继续利用果蝇的遗传易操纵性和分子资源，以进一步从机制上深入了解SPARC在幼虫发育过程中的不同形态调节作用。到目前为止，我们的工作提供了强有力的证据，表明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，以完全接受分泌的胞外蛋白的这一独特功能。这项建议中概述的工作将提供这一信息，并将检验我们的假设，即翼想象盘源性SPARC代表一种不能结合Col(IV)的糖形式，并发挥与脂肪体来源的SPARC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.