Regulation and Function of Phosphoinositide Lipid Signals

细胞器是细胞的“器官”。细胞器有很多种，每一种都有自己独特的生化和功能特性。例如，内质网(ER)是发生蛋白质合成的膜小管的迷宫，而溶酶体是充满消化酶的小而圆的细胞器，可以消除受损的蛋白质和微生物等不需要的物质。细胞器是如何形成或变化的，是细胞生物学中的一个关键问题。磷脂酰肌醇(PIP)脂类是细胞器特性的设计师。有7种PIP在细胞器中有不同的分布。每个PIP物种都结合了一组独特的蛋白质，这些蛋白质用特定的分子特性装饰宿主细胞器。为了理解PIP如何定义细胞器的特性，我们需要了解A)合成和降解PIP的酶是如何被调控的，以及B)PIP及其效应蛋白是如何工作的。磷脂酰肌醇-3，5-二磷酸[PI(3，5)P2]是由脂蛋白激酶PIKfyve合成的一种特异性PIP。PI(3，5)P2控制溶酶体的性质，这对细胞清除不需要的物质很重要。我的NSERC资助的研究重点是了解PIKfyve是如何受到监管的，以及它是如何发挥作用的。我们发现，抑制PIKfyve会损害免疫功能，包括中性粒细胞向微生物的迁移(趋化)和微生物的吞噬和消化(吞噬和吞噬小体成熟)。此外，我们还发现，在PIKfyve消融过程中，溶酶体在融合后未能与其他溶酶体分离(分裂)，从而导致它们的融合。这种分离缺陷的发生可能是因为PIKfyve调节了产生力量的蛋白质机制和/或使导致分裂的膜变形。在NSERC的支持下：i)我们将使用基于溶酶体分离、分子标记和质谱仪的无偏见方法来了解溶酶体的组成在PIKfyve消融过程中是如何变化的。这些数据可能识别与膜分裂有关的复合体。Ii)我们将研究PIKfyve控制吞噬和细胞迁移的机制。我们将使用免疫细胞和癌细胞来测试这一假设，即PIKfyve协调肌动蛋白细胞骨架的组装和拆解，以塑造细胞形状并产生这些过程所需的力。Iii)我们将研究PIKfyve如何调节肌动蛋白在溶酶体上的组装，以及这是否与内质网结合来划分分裂位点。内质网与其他细胞器的接触部位正在成为细胞器分裂的介体。总而言之，这项研究将为PIKfyve如何控制溶酶体的功能以及与细胞的力产生机制的接口提供新的见解。这可能有助于研究人员更好地了解PIKfyve缺失造成的有害影响。反过来，这可能为加拿大制药业提供新的策略来治疗由PI(3，5)P2功能障碍引起的Charcot-Marie Tooth神经退行性疾病。

Organelles are the "organs" of cells. There are many types of organelles, each with their own unique biochemical and functional properties. For example, the endoplasmic reticulum (ER) is a labyrinth of membrane tubules where protein synthesis occurs, whereas lysosomes are small, round organelles packed with digestive enzymes that eliminate unwanted materials like damaged proteins and microbes. How organelles form, or change, is a key question in cell biology. The phosphoinositide (PIP) lipids are architects of organelle identity. There are seven PIP species that are differentially distributed among organelles. Each PIP species binds a unique set of proteins, which decorates the host organelle with specific molecular properties. In order to understand how PIPs define organelle identity, we need to understand A) how the enzymes that synthesize and degrade PIPs are regulated and B) how PIPs and their effector proteins work. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is a specific PIP that is synthesized by the lipid kinase PIKfyve. PI(3,5)P2 governs lysosome properties, which is important for cells to clear unwanted material. My NSERC-funded research has focused on understanding how PIKfyve is regulated and how it functions. We showed that inhibition of PIKfyve impairs immune function, including neutrophil migration towards microbes (chemotaxis) and the engulfment and digestion of microbes (phagocytosis and phagosome maturation). In addition, we discovered that lysosomes fail to separate (fission) from other lysosomes after fusion during PIKfyve ablation, causing their coalescence. This separation defect may occur because PIKfyve modulates protein machinery that generate force and/or deforms membranes that elicit fission. With NSERC support: i)We will use unbiased methods based on lysosome isolation, molecular tagging, and mass spectrometry to understand how the composition of lysosomes change during PIKfyve ablation. These data may identify complexes involved in membrane fission. ii)We will study the mechanisms by which PIKfyve governs phagocytosis and cell migration. We will use immune and cancer cells to test the hypothesis that PIKfyve coordinates the assembly and disassembly of the actin cytoskeleton to mold cell shape and generate forces necessary for these processes. iii)We will study how PIKfyve modulates the assembly of actin on lysosomes and if this interfaces with the ER to demarcate fission sites. ER contact sites with other organelles is emerging as a mediator of organelle fission. In all, this research will provide new insight into how PIKfyve governs lysosome function and interfaces with the cell's force generating machinery. This may then aid researchers better understand deleterious effects caused by PIKfyve loss. In turn, this may provide the Canadian pharmaceutical industry with novel strategies to treat conditions like Charcot-Marie Tooth neurodegenerative disease caused by PI(3,5)P2 malfunction.