POWRE: Mechanisms of Organelle Autophagy in the Yeast S. cerevisiae

9752962罗宾逊 这是POWRE研究增强奖。 在细胞重塑过程中，一个被称为自噬的过程在将多余的细胞质蛋白和细胞器转运到溶酶体进行降解中起作用。 这种细胞重塑发生在分化、营养剥夺、变态、衰老和转化过程中;自噬也会清除可能因各种原因积累的异常细胞器。 因此，自噬是一个非常重要的过程。 在自噬过程中，双膜结构包围或吞噬含有待降解物质的细胞质区域，产生双膜结合的细胞器，即自噬体。 自噬体通过将外膜与溶酶体或空泡膜融合来递送其内容物用于降解。 本项目的目标是揭示细胞器自噬的分子和细胞机制。 实验将在酵母（酿酒酵母）中进行。 这种生物体通常用作细胞生物学研究的模型，因为它为遗传，细胞生物学和分子分析提供了优势。 实验方法将是用绿色荧光蛋白（GFP）标记酵母细胞的过氧化物酶体，并使用数字延时荧光显微镜以及定量荧光法来观察和测量过氧化物酶体自噬。 一些计划中的实验将测试自噬是否可以是一个选择性的过程，其中特定的多余结构选择性地针对自噬途径。 为了做到这一点，过氧化物酶体和线粒体降解率将同时测量的条件下，其中一个细胞器，而不是其他，是细胞活力所需的。 将被测试的第二个假设是，一般的膜运输组件以及细胞的细胞骨架的关键组件在酵母中自噬体的形成，运输或功能中发挥作用。 这些实验将结合联合收割机assa ys细胞器自噬的方法，在酵母中，以具体地抑制特定的蛋白质功能。 这些实验将专门评估参与膜融合的两种蛋白质NSF和Kar 2 p以及酵母细胞骨架系统的两种组分微管和肌动蛋白在自噬中的潜在作用。 最后，将进行遗传筛选以确定参与自噬的新分子。 将使用激活的细胞分选来富集由于延迟或消除的自噬而保留GFP标记的过氧化物酶体的突变细胞。 通过该筛选收集的突变体的表征是理解自噬分子机制的长期目标的一部分，但超出了本奖项的范围。 这个项目代表了主要研究者在职业生涯中的一个重大中断后回到活跃的细胞生物学研究。 来自POWRE奖的支持将使主要研究者能够为一个新颖而有前途的研究途径开发必要的初步结果。 ***

9752962 Robinson This is a POWRE Research Enhancement Award. During cellular remodeling, a process known as autophagy plays a role in transporting superfluous cytoplasmic proteins and organelles to the lysosome for degradation. Such cellular remodeling occurs during differentiation, nutrient deprivation, metamorphosis, aging and transformation; autophagy also removes abnormal organelles that may accumulate for various reasons. Thus, autophagy is a vitally important process. During autophagy, a double membrane structure surrounds or engulfs a region of cytosol containing the material to be degraded, creating a double membrane bound organelle, the autophagosome. Autophagosomes deliver their contents for degradation by fusing the outer membrane with the lysosomal or vacuolar membrane. The objective of this project is to unravel the molecular and cellular mechanisms involved in organelle autophagy. The experiments will be performed in the yeast, Saccharomyces cerevisiae. This organism is commonly used as a model for cell biological studies because of the advantages it offers for genetic, cell biological, and molecular analyses. The experimental approach will be to label the peroxisomes of the yeast cells with green fluorescent protein (GFP) and use digital time-lapse fluorescent microscopy as well as quantitative fluorimetry to observe and measure peroxisome autophagy. Some of the planned experiments will test whether autophagy can be a selective process where specific superfluous structures are selectively targeted to the autophagic pathway. To do this, rates of peroxisome and mitochondrial degradation will be simultaneously measured under conditions where one organelle, but not the other, is required for cell viability. A second hypothesis that will be tested is that general membrane trafficking components as well as key components of the cell's cytoskeleton play a role in the formation, transport or function of the autophagosomes in yeast. These experiments will combine assa ys for organelle autophagy with methods available in yeast to specifically inhibit specific protein functions. The experiments will specifically assess potential roles in autophagy of two proteins involved in membrane fusion, NSF and Kar2p, and two components of the yeast cytoskeletal system, microtubules and actin. Finally, genetic screens will be carried out to identify new molecules involved in autophagy. Fluorescence-activated cell sorting will be used to enrich for mutant cells that retain GFP-labeled peroxisomes due to delayed or abolished autophagy. The characterization of mutants collected by this screen is part of the longer term goal of understanding the molecular mechanisms of autophagy but is beyond the scope of the present award. This project represents a return to active cell biological research for the Principal Investigator, after a major hiatus in her career. The support from a POWRE award will allow the Principal Investigator to develop the necessary preliminary results for a novel and promising avenue of research. ***